Reactive oxygen and nitrogen species (RO/NS) such as nitric oxide (NO), hydroxyl radical (OH·), and superoxide anion (O2−) are generated in a variety of neuropathological processes and damage neurons. In the present study, we investigated the neuroprotective effects of rat astrocytes against RO/NS-induced damage using neuron–glia cocultures, and the effects were compared to those of microglial cells. Sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1), and FeSO4 were used to generate NO, O2− and NO, and OH·, respectively. Solely cultured neurons, which were transiently exposed to these agents, degenerated, possibly through apoptotic mechanisms as revealed by in situ detection of DNA fragmentation, whereas neurons cocultured with either astrocytes or microglial cells were viable even after exposure to RO/NS. In contrast, most neurons cocultured with meningeal fibroblasts degenerated. Astrocyte-conditioned medium partially attenuated RO/NS-induced neuronal damage. When neurons were cultured on astrocyte-derived extracellular matrix (AsECM), neuronal death induced by SNP and FeSO4 was almost completely inhibited. AsECM contained significant amounts of laminin and fibronectin, and pure fibronectin and laminin also protected neurons against RO/NS-induced damage in the same manner as AsECM. These results suggest that astrocytes can protect neurons against RO/NS-induced damage by secreting soluble and insoluble factors. GLIA 28:85–96, 1999. © 1999 Wiley-Liss, Inc.