NATO Secretary-General Anders Fogh Rasmussen says it would be an "historic mistake" if Russia intervened further in Ukraine following the annexation of Crimea.
On April 2, Rasmussen said Russia's military buildup on the Ukrainian border was a "matter of grave concern." Western officials have said tens of thousands of Russian forces have massed in the region.
"These Russian military armed forces are at very high readiness," he said. "This is really a matter of grave concern. If Russia were to intervene further into Ukraine, I wouldn't hesitate to call it an historic mistake."
Rasmussen was speaking after a two-day NATO foreign ministers' meeting in Brussels.
His remarks came one day after the NATO ministers suspended all "practical" civilian and military cooperation with Russia in response to last month's annexation of Crimea.
They also agreed to draft measures to strengthen alliance defenses and reassure eastern NATO members who may feel menaced by Russia's actions in Ukraine.
On April 2, Russian Foreign Minister Sergei Lavrov expressed concern over NATO's decisions to U.S. Secretary of State John Kerry in a telephone conversation
In Washington, State Department spokeswoman Marie Harf said the phone call was brief and that Kerry "conveyed the strong support he was hearing for the people of Ukraine - and the legitimate government of Ukraine - from his counterparts during his NATO meeting in Brussels."
Russian Foreign Ministry spokesman Aleksandr Lukashevich said the NATO statements resembled Cold War-era rhetoric.
"The language of the [NATO] statement sounds more like a verbal snippet from the Cold War era," he said. "It is difficult to imagine who in today's world is going to gain from the curtailment of Russia-NATO interaction, countering such modern threats and international security challenges as terrorism, piracy, natural and human-made disasters. In any case, it definitely won't be Russia, and definitely not the NATO member states."
The head of President Vladimir Putin's administration, Sergei Ivanov, said Russia was concerned about reports of possible military buildups in eastern NATO members.
Earlier on April 2, NATO's top military commander was quoted as saying analysts believe Russia already has all the forces it needs on Ukraine's border if Moscow were to decide to carry out an "incursion" into the country.
NATO's Supreme Allied Commander Europe, U.S. Air Force General Philip Breedlove, described the situation on the border as "incredibly concerning."
He told interviewers from the Reuters news agency and "The Wall Street Journal" that Russia could achieve the military goals of any Ukrainian incursion in between three to five days.
Breedlove said potential targets for Russia included an incursion into southern Ukraine to establish a land corridor to Crimea, pushing beyond Crimea to the port of Odesa, or even threatening to connect to Moldova's pro-Moscow breakaway region Transdniester.
Meanwhile, the Pentagon said on April 2 that it was sending 175 new troops to a Romanian base near the Black Sea.
Spokesman Army Colonel Steve Warren told reporters that the decision was made before Russia seized control of Crimea.
Warren said the United States also intends to soon send a navy ship into the Black Sea "to reassure our allies of our commitment to the region."
"It is a direct result of the current situation in Ukraine," he added.
With reporting by Reuters and Interfax