The cytotoxic effects of hypoxia and excitatory amino acids on cultured retinal ganglion cells were studied. The influence of coculture with retinal Müller glia and cortical astrocytes on cell survival was evaluated, as were the effects of the N-methyl-DL-aspartate inhibitor, MK-801. Dissociated retinal neurons from 7-day-old Sprague-Dawley rats were plated on a laminin substrate, neocortex-derived glial monolayers, or Müller cell monolayers. Ganglion cells were labeled by injection of DiI into the superior colliculus 2 days prior to dissociation. Exposure of cultured ganglion cells to glutamate and N-methyl-DL-aspartate showed a time- and concentration-dependent survival rate. Exposure of cells to hypoxia demonstrated a survival rate that was dependent on time and O2 concentration. Excitotoxic and hypoxic damage was entirely blocked by the specific non-competitive inhibitor of N-methyl-DL-aspartate, MK-801. Retinal ganglion cells cultured on cortical astrocytes and retina-derived Müller glia showed significantly better survival rates (P < 0.001) than cells cultured on laminin-coated dishes under control conditions, in hypoxia (9% to 15% O2), and after exposure to 200 microM glutamate. Retinal ganglion cells cultured on Müller glia showed significantly better survival rates (P < 0.01) than those cultured on cortical astrocytes under conditions of hypoxia (9% to 15% O2) and exposure to 200 microM glutamate. The results demonstrate that excitotoxic and hypoxic damage to cultured retinal ganglion cells is moderated by NMDA receptor blockade and by the presence of glial cells, especially retinal Müller cells. This system may provide a useful model for studying the pathophysiology of excitotoxicity and hypoxia on cultured retinal ganglion cells, and may be used to help identify potentially clinically useful therapeutic agents.