Denis Pushilin, the head of the pro-Russian separatist government in the Donetsk region, released a statement Saturday announcing a full troop mobilisation and urging reservists to show up at military enlistment offices.
The move comes amid a spike in violence along the line of contact between Ukrainian forces and Russia-backed rebels in the region in recent days that fuelled Western fears that Moscow could use it as a pretext for an invasion.
On Friday, separatist authorities in Donetsk and Luhansk announced a mass evacuation of women, children and the elderly to neighbouring Russia.
Shortly after the evacuation effort started, several explosions hit the rebel-controlled areas.
The separatist conflict in eastern Ukraine erupted in 2014 and has killed more than 14,000 people.
US President Joe Biden says he is now "convinced" Russian President Vladimir Putin has decided to invade Ukraine and assault the capital.
After weeks of saying the US was not sure if Putin had made the final decision, Biden said on Friday that his judgement had changed, citing American intelligence.
"As of this moment, I'm convinced he's made the decision," Biden said. "We have reason to believe that." He reiterated the assault could occur in the "coming days".
The president's comments at the White House followed a day of rising violence that included a humanitarian convoy hit by shelling and a car bombing in Donetsk.
Meanwhile, the Kremlin announced massive nuclear drills to flex its military muscle, and Putin pledged to protect Russia's national interests against what it sees as encroaching Western threats.
Biden reiterated his threat of crushing economic and diplomatic sanctions against Russia if it does invade, and pressed Putin to reconsider.
He said the US and its Western allies were more united than ever to ensure Russia pays a steep price for any invasion.
As further indication that the Russians are preparing for a major military push, a US defence official said an estimated 40 to 50 per cent of the ground forces deployed in the vicinity of the Ukrainian border have moved into attack positions closer to the border.
That shift has been under way for about a week, other officials have said, and does not necessarily mean Putin has decided to begin an invasion.
The official also said the number of Russian ground units known as battalion tactical groups in the border area had grown to as many as 125, up from 83 two weeks ago. Each group has 750 to 1000 soldiers.
Lines of communication remain open: The US and Russian defence chiefs spoke on Friday. Secretary of State Antony Blinken and Russian Foreign Minister Sergey Lavrov agreed to meet next week.
But with an estimated 150,000 Russian troops now posted around Ukraine's borders, the long-simmering separatist conflict could provide the spark for a broader attack.
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