Britain has rejected a Spanish proposal to hold one-on-one talks over the sovereignty of Gibraltar.
The move comes after Spain issued an appeal for such negotiations to be held "as soon as possible".
Spanish Foreign Minister José Manuel Garcia-Margallo made the appeal in the Wall Street Journal today.
However, a spokesperson for British Prime Minister David Cameron said it would not enter into any talks on the subject.
The spokesperson said "sovereignty is clear in our minds", before adding that the only talks Britain could envisage would be between Spain and Gibraltar over fishing practices, not over the enclave's sovereignty or control of its fishing waters.
A diplomatic war of words over the rocky Mediterranean outpost began in July after it built an artificial reef in contested waters using concrete blocks. Spain said the reef restricted access for its fishermen.
Spain lays claim to the territory, which has a population of just 30,000, but which it ceded to Britain by treaty 300 years ago.
"Since the beginning of the current legislature, the Spanish government has urged the UK to resume bilateral negotiations as soon as possible on issues of Gibraltar's sovereignty, which have been on hold for too long," wrote Mr Garcia-Margallo.
Spain tightened border controls after the creation of the reef and threatened to take further action, including introducing a €50 levy to cross the border from Gibraltar into Spain.
The European Commission said yesterday it would send a fact-finding mission to Gibraltar to examine the legitimacy of those controls.
Meanwhile, opposition parties in Spain and Gibraltar's Chief Minister Fabian Picardo have accused Spain's ruling centre-right government of using the conflict as a smokescreen for a corruption scandal involving senior politicians.
Mr Garcia-Margallo reiterated Prime Minister Mariano Rajoy's demand for Britain to remove the blocks used for the reef.
He said Spain was also open to hosting meetings on the topic between other authorities, such as the government of Gibraltar and the local government of neighbouring Spanish region Andalusia.
"The dialogue must be bilateral and respect international, European and national laws," he said.
However, the British spokesperson made it clear any talks would be limited in scope.
"There needs to be some dialogue on fishing, but that would not be about who's responsible for the waters as that's clear."
Another British spokesperson said yesterday that Spain had built similar reefs along its own coastline.
Long queues have formed at the border in recent weeks as the rift over the reef has escalated.
Gibraltar is not part of the Schengen open border agreement between some EU member states, meaning Spain is entitled to carry out proportionate checks.
Mr Garcia-Margallo highlighted Spain's concerns about the British territory, including Gibraltar's opaque tax regime and said Spain had initiated proceedings to denounce bunkering, or refuelling ships at sea.
The minister said smuggling was also a worry as European Union excise duties do not apply on the rocky outcrop.
Spain has threatened to take its claim on Gibraltar to the United Nations.