Survival of dissociated 19-day fetal rat telencephalic neurons in a hormone-free defined medium required the addition of insulin at pharmacological concentrations. However, survival of astrocytes cultured from the cerebral cortex of newborn rats in the same medium did not require insulin. When fetal neurons were incubated with astrocyte conditioned medium or plated on a monolayer of astrocytes, their survival was significantly increased in the absence of insulin. This effect of astrocyte conditioned medium was visibly inhibited by affinity chromatography on an anti-insulin protein A agarose column. A 5-30 kDa ultrafiltration fraction of astrocyte conditioned medium also increased neuronal survival. In addition, the 5-30 kDa fraction stimulated [3H]leucine incorporation into the TCA insoluble material from cultured neurons and competed for [125I]insulin binding to intact neuronal cultures. These results indicate that cultured astrocytes produce a factor with biological and immunological properties similar to those of insulin. This factor may in part mediate the observed neurotrophic effects of astrocyte conditioned medium and may play a role in the normal development and differentiation of central nervous system neurons. © 1988.