AMPA/kainate receptor-mediated excitotoxicity is believed to play a pathogenic role in amyotrophic lateral sclerosis. To further characterize the mechanisms involved in AMPA/kainate receptor-mediated motoneuron injury, we investigated the influence of spinal glial cells on kainate-induced motoneuron death in vitro. A motoneuron-enriched neuronal population was obtained from embryonic mouse spinal cord by metrizamide density centrifugation. This population was cultured either on a pre-established glial feeder layer of ventral spinal origin (coculture) or in glia-free conditions (monoculture). Glial feeder layers significantly enhanced basal survival of neurons, and supported neuronal differentiation as judged by neuronal morphology and expression of the motoneuron markers peripherin and SMI-32. Neuronal vulnerability to kainate was two- to three-fold higher in coculture than in monoculture, and increased significantly with time in coculture. The effects of glial feeder layers on neuronal basal survival, differentiation and kainate vulnerability were not mimicked by conditioned medium from glial cells. The increase in neuronal kainate vulnerability with time in coculture was associated with a marked rise in the proportion of cocultured neurons possessing Ca2+-permeable AMPA/kainate receptors, as determined by kainate-activated Co2+-uptake. Neurons in monoculture were unstained by kainate-activated Co2+-uptake. Neurons were immunoreactive to specific antibodies against the AMPA receptor subunits GluR1 and GluR2 both in monoculture and coculture. This study indicates that motoneuron differentiation in coculture is associated with increased vulnerability to kainate and increased expression of Ca2+-permeable AMPA/kainate receptors. In this paradigm glial cells support basal survival and differentiation of neurons, but potentiate kainate-induced neuronal death.