By Jenny Gross 

LONDON--Prime Minister David Cameron's Conservative Party suffered a significant loss to a small but growing anti-European Union party, underscoring the challenge he faces at the coming general election and the growing debate in the U.K. about its relationship with Europe.

The U.K. Independence Party, which advocates Britain's departure from the EU, secured a roughly 7% lead over the Conservatives to secure its second seat in Britain's parliament. That came despite Mr. Cameron throwing himself behind the campaign to retain the seat in Rochester and Strood, 30 miles southeast of London, where Conservative lawmaker Mark Reckless resigned from the party to contest the seat under UKIP's banner.

The results are a blow to Mr. Cameron six months before what is expected to be a close-fought general election. Both of UKIP's recent wins were made by previously Conservative members of parliament who had stepped down in recent months to contest their seats as UKIP candidates.

While UKIP remains a small party, it poses an electoral threat to the Conservatives because it could drain away votes in closely contested constituencies.

The result underscores the increasingly fragmented nature of the British political landscape, which has long been dominated by the Conservatives and Labour. With many analysts expecting a hung parliament in May's election, some say there could be a situation where neither is able to form a coalition with just one other party.

The Rochester result was also a disappointment for the two other major parties, with both Labour and the Liberal Democrats, who are the junior partner to the Conservatives in the coalition government, losing votes. The Lib Dems, traditionally the U.K.'s third largest party, came in fifth behind the Green Party. Opinion polls now show UKIP as the third most popular party, behind the Conservatives and Labour.

Some analysts have linked the fragmentation, which is also being seen by mainstream parties across Europe, to a growing sense of frustration with the EU that has been fueled by the financial crisis. How UKIP fares as the British economy continues to recover will be an important indicator in whether the changes are temporary or mark a fundamental shift in U.K. politics.

For its part, UKIP hailed the results as transformative. UKIP leader Nigel Farage and Mr. Reckless said the victory in Rochester, which the party had listed as 271 on its list of winnable seats, provides a major boost to its prospects for the general election. Mr. Reckless secured 16,867 votes, or a 42% share of ballots cast.

While the win bolsters UKIP's presence in parliament, the party could find it more difficult to retain the seat in the general election as voters may be less likely to vote for a fringe party that they know won't win a majority.

"I'm absolutely determined to win this seat back at the next general election," Mr. Cameron said Friday, speaking to the British Broadcasting Corporation. William Hague, the Conservative Leader of the House of Commons, told the BBC that his party must "listen carefully to what people are saying to us." Mr. Cameron and his government colleagues have been trying to refocus the political debate on the government's handling of the economy, which is performing well compared with other Western countries and is traditionally seen as a Conservative strong suit.

Still, the Rochester result is likely to increase pressure on Mr. Cameron to toughen his stance on the EU and immigration, key planks of UKIP's message. Mr. Cameron is expected to give an update in the coming weeks on how he plans to curb immigration from within the EU, which accounts for a significant portion of inflows into the U.K. But other EU leaders have warned the British prime minister against trying to block the free movement of people, which they see as a fundamental principle of the 28-country bloc.

UKIP's growing popularity underscores the intensifying debate in the U.K. about its membership of the EU. Mr. Cameron has promised to renegotiate Britain's relationship with Brussels and--if he wins--hold a referendum by 2017 on whether the U.K. should remain in the EU. Mr. Cameron has said he wants the U.K. to remain within a reformed EU.

"We believe that Brexit is unlikely but not impossible in the next five years," said Citigroup Inc. in a an analyst report published Friday. "The rise in support for UKIP has not gone alongside a rise in support for EU exit. However, perhaps uniquely among EU countries, the possibility of EU exit is not implausible for the U.K.," Mr. Saunders wrote. "This risk is likely to weigh on business confidence, especially among foreign-owned firms, in coming years."

Write to Jenny Gross at jenny.gross@wsj.com

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