A cytotoxic effect, as measured by 51Cr release, was detected after a 6-h incubation with two strains of Borrelia burgdorferi with neonatal rat primary brain cultures and with astroglial enriched cultures derived from the primary rat brain cells. A low-passage strain, J31, induced a significantly greater cytotoxic effect than did strain B31 in long-term in vitro culture. Live spirochetes and sonicates of both strains induced cytotoxicity, whereas heat-killed organisms did not. The degree of injury was greater in the primary brain than in the astroglial enriched cultures. Scanning electron microscopy revealed marked contraction of the membrane sheets and bleb production by the oligodendroglia of primary brain cultures after incubation with B. burgdorferi. The astroglial layer appeared unharmed.