Gibraltar accused Spain of causing long traffic jams with tightened border controls, saying it was "clearly a response" to rising political tensions over the British territory.
As Gibraltar emerges as a sore point in Britain's exit negotiations with the European Union, Deputy Chief Minister Joseph Garcia complained of traffic tie-ups on Wednesday on the border of the rocky British outcrop at Spain's southern tip.
"The latest action of Spain is obviously and clearly a response to the latest political climate," Garcia told broadcaster GBC. "It is what they've always done but certainly it is totally and absolutely unacceptable."
"We have been told that the police officers deployed at the border, the Policia Nacional, are not the ones that are here normally. They don't quite understand how they need to conduct the checks at the border," he added.
Police in the territory known as The Rock, which is home to 32,000 people, tweeted: "All those driving toward Spain should expect long delays."
Gibraltar's government earlier posted on Twitter that vehicles faced two-hour lines to cross into Spain.
Neither Spain's interior ministry nor the national police responded to AFP's requests to confirm that border measures had been tightened.
Some 10,000 people cross from Spain to Gibraltar to work every day, with the outcrop dependent on the small land border for trade and tourism.
"Spain has used traffic jams as a political weapon against Gibraltar since the day the border opened," Garcia charged.
He noted that there were similar scenes at the frontier in 2013 when, in the midst of a diplomatic row, Madrid doubled its border controls until the European Commission stepped in to calm the situation.
Tensions soared last week when the European Union said Spain should have a veto on extending any trade deal to Gibraltar after Britain leaves the bloc.
London and Madrid have had a long and bitter dispute over Gibraltar, which has been a British territory for more than 300 years.
Fearing that Madrid is seeking to take advantage of Brexit to impose its control over the enclave, Gibraltar reacted angrily to the EU move and London firmly expressed its support for the territory.
A Spanish warship also sailed into disputed waters off Gibraltar on Tuesday, raising tensions further, although such incidents are not uncommon. The European Parliament on Wednesday overwhelmingly adopted tough "red lines" for negotiations over a Brexit deal, on which EU lawmakers will have the final say in two years' time, but omitted any mention of the flashpoint issue of Gibraltar.