Spanish prime minister Mariano Rajoy has said the country will take "all legal measures" to protect its interests in Gibraltar, it has been reported.
Downing Street said the British position on the issue "has been quite clear" and the Foreign Office (FCO) said it was aware of Mr Rajoy's comments.
The Spanish PM's intervention came two days after a phone call with David Cameron, who told his counterpart the situation at the border with Gibraltar was "not acceptable".
Number 10 said that in the "constructive" phone call Mr Rajoy agreed to reducing measures at the border which have led to lengthy delays for Gibraltarians, but a statement issued by the Spanish government afterwards made no reference to any such concession and insisted procedures at the frontier were proportionate.
The Prime Minister said he was very clear that Britain "will always stand up for the people of Gibraltar".
His intervention followed a formal protest by Britain's ambassador in Madrid over "disproportionate" checks at the border and Spanish threats to levy a charge on vehicles crossing the border and to close airspace.
The dispute has flared up over allegations of damage to fishing grounds caused by Gibraltarian authorities following the creation of an artificial reef. Gibraltar's chief minister, Fabian Picardo, has likened the Spanish behaviour to something from the Franco era or the regime in North Korea.
The European Commission has suggested organising a "technical meeting" with the Spanish authorities about the border controls in September or October.
A Commission spokesman said that because Gibraltar, like the UK, was not part of the Schengen open borders area in Europe "checks on persons can be carried out at its border with Spain" but that controls must be proportionate.
The Ministry of Defence (MoD) announced on Thursday that a British warship will visit Gibraltar later this month.
HMS Westminster and two support ships will visit Gibraltar while other elements of a task force will visit Spanish ports as part of training exercises known as Cougar 13.
The MoD said the deployment is "long-planned" and not connected to the political tensions in the region. Cougar 13 is an annual exercise designed to hone skills and the deployment will also see port visits in Portugal and throughout the Mediterranean.
Spain claims sovereignty over Gibraltar, which stands on the southernmost tip of the Iberian peninsula but has been a British Overseas Territory since the Treaty of Utrecht in 1713. The UK Government has made clear that it will not negotiate over sovereignty as long as Gibraltar's people want to remain British.
Why are you making commenting on The Herald only available to subscribers?
It should have been a safe space for informed debate, somewhere for readers to discuss issues around the biggest stories of the day, but all too often the below the line comments on most websites have become bogged down by off-topic discussions and abuse.
heraldscotland.com is tackling this problem by allowing only subscribers to comment.
We are doing this to improve the experience for our loyal readers and we believe it will reduce the ability of trolls and troublemakers, who occasionally find their way onto our site, to abuse our journalists and readers. We also hope it will help the comments section fulfil its promise as a part of Scotland's conversation with itself.
We are lucky at The Herald. We are read by an informed, educated readership who can add their knowledge and insights to our stories.
That is invaluable.
We are making the subscriber-only change to support our valued readers, who tell us they don't want the site cluttered up with irrelevant comments, untruths and abuse.
In the past, the journalist’s job was to collect and distribute information to the audience. Technology means that readers can shape a discussion. We look forward to hearing from you on heraldscotland.com
Comments & Moderation
Readers’ comments: You are personally liable for the content of any comments you upload to this website, so please act responsibly. We do not pre-moderate or monitor readers’ comments appearing on our websites, but we do post-moderate in response to complaints we receive or otherwise when a potential problem comes to our attention. You can make a complaint by using the ‘report this post’ link . We may then apply our discretion under the user terms to amend or delete comments.
Post moderation is undertaken full-time 9am-6pm on weekdays, and on a part-time basis outwith those hours.
Read the rules hereComments are closed on this article