President Biden warned Russian President Vladimir Putin that the U.S. and its allies would meet a military invasion of Ukraine with strong economic penalties, moves to bolster Ukrainian defenses and fortify support for Eastern European nations.

For two hours Tuesday, the two leaders held a secure video call to address what the U.S. has described as large and unusual troop movements near Russia’s border with Ukraine in recent weeks.

Mr. Biden reaffirmed U.S. support for Ukraine’s sovereignty and urged Mr. Putin to return to diplomatic talks to resolve the conflict in Ukraine’s Donbas region, where Kyiv is fighting Russian-backed separatists, White House national security adviser Jake Sullivan said following the call. The two presidents, he said, also discussed strategic stability, ransomware and Iran, and assigned staff to follow up on the call.

Mr. Sullivan described the conversation as direct and straightforward. There was “a lot of give and take. No finger wagging. But the president was crystal clear about where the United States stands on all of these issues,” he said.

Yury Ushakov, a presidential aide to Mr. Putin, characterized the talks as frank and businesslike in tone. At the same time “there was a place for jokes, for the exchange of compliments,” he said.

The Kremlin said the talks focused on the Ukrainian crisis and the lack of progress in the 2015 diplomatic talks, known as the Minsk process, aimed at securing a peaceful settlement in Donbas. Mr. Putin expressed concern about what Moscow sees as provocative actions by Kyiv against Donbas and accused Ukraine of sabotaging the peace process, Mr. Ushakov said.

Mr. Biden has been working in tandem with members of NATO to defuse the increasingly tense developments in Eastern Europe. A Russian invasion of Ukraine would be one of the most significant military actions in Europe since the Cold War’s end, posing a security challenge for the European Union, Western European powers and NATO.