We isolated motoneurons from E15 dissociated mouse spinal cord by density centrifugation and planted them onto poly-ornithine-coated coverslips in a growth medium (DMEM/F12) supplemented with progesterone, transferrin, selenium, horse serum and muscle extract. Under these conditions only 28% of the motoneurons survived for 8 days. When living astrocytes on a separate coverslip were introduced into dishes containing motoneurons, there was a two-fold increase in neuronal survival. The addition of insulin and insulin-like growth factor I (IGF-I) to such cultures alone or together, still further increased motoneuron survival, but this did not happen in the absence of astrocytes. We conclude that (a) astrocytes exert a trophic role in the survival of spinal motoneurons, (b) the effect does not require physical contact of the cells, and (c) insulin and IGF-1 have neurotrophic activity for motoneurons, an effect possibly mediated by living astrocytes.