Tensions between Britain and Spain are still running high as warships head for a training exercise near Gibraltar.
HMS Westminster left Portsmouth amid reports that tough checks on the Spanish side of the border are causing three-hour delays.
Downing Street has signalled that it is preparing a legal challenge to the "politically motivated" controls.
There has been a suggestion that a 50 euro (£43.30) fee could be imposed on every vehicle entering or leaving the British overseas territory through the fenced border.
Type 23 frigate HMS Westminster will join helicopter carrier HMS Illustrious and the Navy flagship HMS Bulwark.
Other UK ships taking part in the long-planned Cougar '13 deployment are another type 23 frigate, HMS Montrose, and six Royal Fleet Auxiliary (RFA) ships.
It is the third time the exercise has taken place, with the aim of demonstrating the ability to operate an effective maritime force anywhere in the world to protect UK interests.
The Royal Gibraltar Police said queues to enter Gibraltar had been around three hours long, while there were no delays leaving.
The Prime Minister's spokesman has made clear that the legal action would be an "unprecedented step".
The European Commission plans to send a team of investigators to the Gibraltar border in the next couple of weeks to observe the new controls, following complaints from several MEPs and EU citizens about long waits there.