Gibraltar's chief minister has raised concerns after two men fishing off the coast of the British overseas territory complained that the crew of a Spanish customs boat had "fired in their direction".
Fabian Picardo tonight said investigations had confirmed that shots were fired when the men's pleasure boat was "well inside" British Gibraltar territorial waters on Saturday.
He said the "illegal incursion" represented an "extremely serious escalation" of "repeated Spanish violations of British-Gibraltar sovereignty".
And he said the Gibraltar government was "working closely" with the UK Government so that "necessary and appropriate action" was taken to counter "this unacceptable threat".
Mr Picardo outlined his concerns in a statement released by the Gibraltar government.
The Foreign Office said it had lodged a protest.
"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," said a Foreign Office spokeswoman.
"We take seriously any reports of attempts by Spanish law enforcement agencies to interfere with vessels in British Gibraltar territorial waters and threaten or intimidate their crews.
"We continue to work closely with HM Government of Gibraltar and the Royal Gibraltar Police (RGP) to establish the facts surrounding the incident."
A Gibraltar government spokesman said in the statement that the two local men had complained to police.
"On Saturday afternoon, two men on board a Gibraltar-registered pleasure boat reported to the Royal Gibraltar Police (RGP) that, whilst fishing well inside British Gibraltar territorial waters, four pistol shots had been fired in their direction by the crew of the Spanish Customs vessel," said the spokesman.
"The Spanish vessel had first ordered the pleasure boat to stop its engines but when this was not done, (it) circled the pleasure boat.
"Its crew also attempted to board the pleasure boat which took evasive action.
"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.
"After the boat contacted the RGP, the police launch, Sir Adrian Johns, which was in the area, quickly made its way to the scene.
"An RGP officer boarded the boat when they got to it and escorted it to the Gibraltar Government Marine Station at Gun Wharf.
"The RGP continue to investigate the incident."
He added: "The two local men have now met with and spoken at length to the Chief Minister and the Deputy Chief Minister and described the details of the whole incident.
"They have fully co-operated with the RGP.
"There is no suspicion that they were doing anything illegal. They had all necessary fishing permits."
Mr Picardo said it was the third incident involving a Spanish customs vessel - known as an SVA - in as many weeks.
"This illegal incursion by the SVA into British Gibraltar territorial waters represents an extremely serious escalation of the repeated Spanish violations of British-Gibraltar sovereignty," he said.
"There is no 'law enforcement' excuse this time.
"Investigations confirm that shots were fired by the SVA at the innocent Gibraltarian pleasure boat.
"The Spanish authorities have, therefore, put the lives of innocent Gibraltarian civilians at risk.
"These actions are criminal offences in Gibraltar.
"Solely by being in the area, the SVA boat was in violation of treaties and the international law of the sea.
"Her Majesty's Government of Gibraltar will not tolerate, under any circumstances, the intimidation or harassment of Gibraltar vessels in British Gibraltar territorial waters.
"We are working closely together with the UK Government so that necessary and appropriate action is taken to counter this unacceptable threat."
He added: "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.
"Proper co-operation remains our aim. The sooner the Spanish authorities adopt a similar co-operative posture the better."
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.
Ministers said repeated incursions were "completely unacceptable and unlawful under the international law" and pledged to urgently raise the matter with the Spanish.
A row broke out after Spanish state vessels chasing suspected drug smugglers made forays into waters around Gibraltar.