Britain is demanding an explanation from Spain over what it called a serious infringement of diplomatic protocol after it was reported that Spanish officials searched a diplomatic bag at the border with Gibraltar.

"As far as we are concerned there is no justification for this infringement of the UK's rights under the Vienna convention," said the Foreign and Commonwealth Office (FCO).

"Diplomatic bags are inviolable. We take very seriously any reported abuse of the protocol surrounding use of the diplomatic bag."

According to the Gibraltar Chronicle, Spanish Guardia Civil officers searched a sealed diplomatic bag as it was being taken from Gibraltar to Spain by a courier last Friday.

The contents of the bag are not known, nor why it was being taken overland across the border. The Guardia Civil refused to comment, or to confirm or deny the incident.

As diplomatic bags are used to carry official documents and other items between governments and their diplomatic missions, the Vienna Convention on Diplomatic Relations stipulates that they must not be opened or seized by unauthorised third parties. It is unclear why Spanish authorities decided to search the bag.

Britain sent a formal note of protest to the Spanish authorities on Tuesday, the FCO said. "The British embassy in Madrid has formally protested to the Spanish Ministry of Foreign Affairs via Note Verbale and we await an urgent explanation," said the FCO. "We will consider further action in light of that explanation.

The FCO said the governor's office in Gibraltar had been sending diplomatic bags for more than 20 years and had no other incident on record in which a fellow EU member state and Nato ally had opened UK diplomatic bags. The last time a UK diplomatic bag was opened was in Zimbabwe in March 2000.

The incident is the latest in a diplomatic dispute in which relations between Madrid and London have sunk to their lowest point in years.

The border between the two countries has been a sore point since the conflict flared up this summer when Gibraltar created an artificial reef off its coast, angering Spanish fishermen.

In a move many saw as retaliatory, Spain tightened controls at the border, leading to queues of up to eight hours for people trying to enter Gibraltar. Spanish authorities said the stricter border controls were necessary as part of a crackdown on tobacco smuggling.