The Foreign Office lodged a formal complaint with Spain after a British diplomatic bag was opened by a Spanish border guard.
Gibraltar's chief minister is to meet David Cameron amid allegations that Spanish border police have come under attack from angry motorists.
Pictures posted on Twitter show Spanish divers hanging flags over the controversial boulders.
This is the third time the Spanish Ambassador has been publicly summoned in relation to Gibraltar since the current Spanish government took office in December 2011.
An MP raised the issue of the alleged incursion by a Spanish vessel into British waters in a Westminster Hall debate earlier today, describing it as "a very serious incident".
Labour MP Jim Dobbin asked: "Is a minister from the Foreign Office coming to make a statement on what is a very, very serious incident?"
Deputy Speaker Lindsay Hoyle replied: "I've been given no notification of a statement to be made but I'm sure the Foreign Office is listening very carefully to what you've just said on such a serious incident."
This video is believed to show a Spanish vessel - the RV Ramon Margalef - in British waters off the coast of Gibraltar.
It was uploaded to YouTube yesterday and purportedly includes extracts of the radio conversation between the ship's skipper and Gibraltar authorities.
ITV News cannot independently confirm the veracity of this footage.
The Foreign Office has summoned Spain's ambassador to the UK over concerns regarding an "ongoing incuresion" by a Spanish vessel into Gibraltar waters.
The Minister for Europe, David Lidington, said:
Despite repeated diplomatic protests to Spain in relation to incursions into British Gibraltar Territorial Waters in recent months, a Spanish State research vessel, the RV Ramon Margalef, undertook significant surveying activity in British Gibraltar Territorial Waters on 18 and 19 November for over 20 hours. When challenged by radio, the vessel responded that it was conducting survey work with the permission of the Spanish authorities and in the interests of the European Community ...
– David Lidington, Minister for Europe
I strongly condemn this provocative incursion and urge the Spanish government to ensure that it is not repeated. We stand ready to do whatever is required to protect Gibraltar’s sovereignty, economy and security.
A Government spokesman said Spain is still acting "unlawfully" at the border with Gibraltar by imposing "disproportionate" checks:
– UK Government spokesman
We remain confident that the Spanish government has acted – and continues to act – unlawfully, through introducing disproportionate and politically motivated checks at the Gibraltar-Spain border.
The fact that the Commission has not found evidence that EU law has been infringed is not the same as concluding that Spain has not acted unlawfully.
The border operated significantly more smoothly than normal during the Commission visit.
Britain has said Spain is still acting illegally by imposing "politically motivated" checks at its border with Gibraltar.
Many residents of Gibraltar reacted angrily to the news that Spain has been cleared of wrongdoing over stringent border controls that caused long traffic queues.
What a farce! You told Spain you were coming, of course you do not find any issues. @eu_commission an absolute joke!
The European Commission has urged UK and Spanish authorities to work together and "strengthen dialogue" at the Gibraltar border.
An EU Commission statement suggested "daily cooperation between the authorities working on each side of the border" to help combat tobacco smuggling.
Commuters faced delays of several hours over the summer caused by stringent checks imposed by the Spanish during a diplomatic row over an artificial reef sunk off Gibraltar.
The European Commission said Spain should "optimise" its profiling system at the Gibraltar border "in order to reduce the large amount of random border controls".
However, the EU said the Spanish authorities had not broken any EU laws with its tight border checks.
"The Commission has not found evidence to conclude that the checks on persons and goods as operated by the Spanish authorities at the crossing point ... have infringed the relevant provisions of Union law," it said in a statement.
The EU Commission has said it has found no evidence that checks by Spanish authorities at the Gibraltar border broke European law.