Britain has accused Madrid of a "serious infringement" of international protocol, saying police had opened a diplomatic bag on the border of Gibraltar.
Madrid denied that its officers had opened any diplomatic bag, which typically are used to carry official correspondence.
Spain lays claim to Gibraltar, a small rocky outcrop on its southern coast that it ceded to Britain 300 years ago, and tensions have run high in recent months.
Britain's Foreign Office said the incident happened last Friday and marked the first time a fellow European Union member and Nato ally had broken international rules by opening a diplomatic bag. The last time such an incident occurred was in Zimbabwe in 2000, it said.
"We take very seriously any reported abuse of the protocol surrounding official correspondence and the diplomatic bag," the Foreign Office said.
"We have asked the Spanish authorities to ensure it does not happen again."