The Foreign and Commonwealth Office (FCO) has raised "serious concerns" after a Spanish Navy vessel wrongly claimed commercial ships off Gibraltar were in Spanish waters.
Spanish Ambassador Federico Trillo has been summoned to appear at the Foreign Office following the incident, which occurred in international waters.
The FCO says the Spanish vessel's actions constitute a breach of the UN Convention on the Law of the Sea.
Minister for Europe, the Rt Hon David Lidington MP, said: “I am extremely concerned by the actions of a Spanish Navy vessel which sought to redirect two commercial vessels heading to and from the Port of Gibraltar, wrongly claiming they were in Spanish waters.
“I condemn this provocative activity and urge the Spanish government to ensure that it is not repeated. Her Majesty’s Government will continue to take whatever action we consider necessary to uphold the interests of Gibraltar, its people, its security and economy."
Prime Minister David Cameron raised Gibraltar with Spanish Prime Minister Mariano Rajoy in the margins of an EU Council meeting.
He made it clear there must not be a repeat of last summer's disruption when the imposition of additional border checks by the Spanish led to long delays at the frontier.
People in Gibraltar feel "under siege" due to the territory being the subject of diplomatic wrangling between the UK and Spanish governments, the House of Commons' Foreign Affairs Committee has said.
"The combination of direct pressure on its border and sovereign waters as well as
diplomatic pressure at the EU, UN and NATO, have resulted in a feeling in Gibraltar of
being under siege," the cross-party committee's latest report says.
The committee accuses Spain of "aggressive policies" towards Gibraltar and says the Government "has responsibilities toward Gibraltar and cannot ignore actions by Spain that are intended to make the lives of Gibraltarians difficult".
The Government has rejected the suggestion from a committee of MPs that it needs to "get off the fence" and act more vigorously to protect British interests in Gibraltar.
A Government spokesman said: "This Government has never been on the fence when it comes to Gibraltar."
"We've been absolutely clear that we will never enter into arrangements under which the people of Gibraltar would pass under the sovereignty of another state against their wishes. And we protest each and every illegal incursion by Spanish state vessels into Gibraltar territorial waters."
A cross-party committee of MPs has called on the Government to threaten Spain with legal action over its treatment of Gibraltar.
The Foreign Affairs Committee also called for a "more robust" response to incursions into British territorial waters and urged ministers to "get off the fence".
The committee said ministers should now set out "naval and police options" to stop Spanish vessels entering British waters, as well as threatening to take Spain to the European Court for breaching EU treaty obligations.
The Spanish ships which entered British waters off Gibraltar acted in an "unlawful" and "dangerous" way, the Foreign Office has said.
"Not only were the actions of the survey vessel unlawful, but it was accompanied by a Spanish Guardia Civil vessel whose dangerous manoeuvring presented a significant safety concern on the waters," Europe minister David Lidington said.
The incursion by a Spanish ship into British waters off Gibraltar is "provocative", "unlawful" and "a violation of our sovereignty", the Europe minister David Lidington has said.
David Lidington was speaking after a Spanish state research trip entered British waters along with a vessel belonging to the Guardia Civil, the part of Spain's police force responsible for protecting the coastline.
According to the United Nations Convention on the Law of the Sea, the waters around Gibraltar are indisputably British territorial waters, under United Kingdom sovereignty, in which only the United Kingdom has the right to exercise jurisdiction.
Her Majesty's Government takes a grave view of any attempt by Spain to exert authority or control within British Gibraltar territorial waters and considers such incursions as a violation of our sovereignty.
I strongly condemn this provocative incursion and urge the Spanish government to ensure that it is not repeated.
The Foreign Office has summoned the Spanish ambassador after a "serious Gibraltar incursion", Reuters reports.
Gibraltar's Chief Minister has expressed his pride at the draw for Euro 2016 after the British overseas territory was forced to switch groups following its brief pairing with Spain.
The blind draw in Nice, France saw the territory, which will compete in qualifying for the very first time, briefly drawn in the same group with Spain before they were moved for "political reasons".
Fabian Picardo, tweeted:
Exciting day. Exciting draw. Proud of GFA's involvement in Euro16. Love the group we are in. Can't wait for the games. All of them!
Gibraltar will now face Scotland, the Republic of Ireland, Germany, Poland and Georgia in Group D when fixtures begin in September.
For the latest football news visit ITV Sport
Gibraltar has been moved after it was selected to be in a group with Spain during the Euro 2016 football tournament draw.
The blind draw in Nice, France, briefly saw the British overseas territory paired up with the current European and world champions in Group C.
But European football's governing body UEFA had already announced in December that Gibraltar and Spain would be kept apart amid continuing political tensions over the territory's sovereignty.
Gibraltar was switched to Group D, where they will face Scotland and the Republic of Ireland among others.
See more on the draw from ITV Sport
A Spanish warship caused a training exercise by British forces to be suspended after entering the waters around Gibraltar.
The Gibraltar Squadron's patrol boat HMS Sabre shadowed the ship, which is said to have used a fake name and call sign, the Gibraltar Chronicle reports.
Royal Navy special forces personnel are believed to have been carrying out a parachute jump at the time, the newspaper said.
Warnings were made to the vessel, which only left the area after radioing back that it was in Spanish waters.
In November, the Foreign Office condemned an incident in which a British diplomatic bag was opened and searched by Spanish Guardia Civil officers on Gibraltar's border with Spain.