Stoltenberg said NATO saw movement of troops, equipment, tanks, artillery and also of advanced air defense systems in violation of a ceasefire agreement.
Russia continuously denies providing arms or troops to support a separatist pro-Russian rebellion in eastern Ukraine, which began after the removal of a Kremlin-oriented Ukrainian president by mass protests in February.
A ceasefire was agreed in early September, but fighting flared again recently.
Stoltenberg told reporters as he arrived for a meeting with European Union defense ministers he had information on a buildup inside Ukraine.
"But we also see a military buildup on the Russian side of the border...This is a serious military buildup and we call on Russia to pull back its troops," he said.
Russia denied similar accusations last week by NATO's top military commander, U.S. Air Force General Philip Breedlove, who said NATO had spotted military equipment arriving from Russia in regions of east Ukraine held by pro-Russian separatist rebels.
General-Major Igor Konashenkov, a Russian Defense Ministry official, dismissed Breedlove's comments last week as anti-Russian "hot air".
Source: Yahoo News