Claims that Spanish customs officials fired shots near a Gibraltarian fishing boat has sparked heightened tensions in the region.
The Foreign Office said it had raised the issue with the Spanish government after two men reported four shots were fired in their direction in waters off the British overseas territory.
It is the third reported incident involving a Spanish customs vessel in as many weeks and prompted a warning that any further incursions would be "actively challenged".
Gibraltar's chief minister Fabian Picardo said the boat had been well inside British Gibraltar territorial waters and the "illegal incursion" represented an "extremely serious escalation" of repeated Spanish violations of British-Gibraltar sovereignty.
The two men complained to police following the incident on Saturday.
They told officials the Spanish vessel ordered them to stop their engines and, when they did not, the boat circled them and attempted to board their vessel.
"During this time, the shots were fired by the (Spanish vessel's) crew into the sea near the boat and objects which they believe to have been bricks were allegedly thrown at the local men," a Gibraltar government spokesman said.
The fishing boat was escorted back to the Gibraltar Government Marine Station after they contacted the Royal Gibraltar Police.
The Gibraltarian government said there was no suggestion the fishermen had been doing anything illegal.
Mr Picardo said the actions had put innocent lives at risk and were criminal offences.
He said the Gibraltarian government was working closely with the UK government to ensure "necessary and appropriate action" is taken to counter "this unacceptable threat".
"Her Majesty's Government of Gibraltar will not tolerate, under any circumstances, the intimidation or harassment of Gibraltar vessels in British Gibraltar territorial waters," he said.
"For our part, the Government of Gibraltar has today changed the posture of Her Majesty's Customs Department at sea such that any future SVA incursions are actively challenged by vessels of HM Customs, who are their counterparts in Gibraltar."
A Foreign Office spokesman said any reported attempt by Spanish law enforcement to interfere with vessels in British Gibraltar territorial waters was taken seriously.
"We have protested this incursion to the Spanish government at a high level and have also raised our extreme concern over the reported interaction between the Spanish vessel and a Gibraltarian pleasure craft during the incursion," the spokesman added.
Earlier this month the UK Government accused Spain of "a clear violation of UK sovereignty" after complaining that boats and helicopters had been sent into British waters in Gibraltar.