"Russia must remove its vehicles and its personnel from the territory of Ukraine immediately. Failure to do so will result in additional costs and isolation," Pentagon spokesman Rear Admiral John Kirby told reporters.
He called the convoy a violation of Ukraine's "sovereignty and territorial integrity by Russia" and questioned if the convoy was on a humanitarian mission.
"Russia should not send vehicles, persons, or cargo of any kind into Ukraine, whether under the guise of humanitarian convoys or any other pretext, without Kiev's express permission," he said.
"We don't have a perfect picture of what's in those trucks," Kirby said.
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Local officials in Ukraine said 20 trucks rolled into the pro-Moscow rebel bastion of Lugansk today, despite objections from Kiev.
The move came as the UN Security Council planned to hold emergency consultations on the convoy.
Lithuania requested the meeting for later today and described the convoy as an "invasion.