Astrocytes have been reported to play a neuropathogenic role within the brain, although little is known about the mechanism underlying astrocyte‐mediated neuronal injury. We investigated the hypothesis that cytokine‐stimulated astrocytes adversely affect neuronal cell survival via generation of the free radical nitric oxide (NO). Primary human astrocytes produced substantial amounts of NO in response to interleukin (IL)‐1α or IL‐1β, which was blocked by the NO synthase inhibitor N^G‐monomethyl‐L‐arginine (NMMA). IL‐1β‐induced NO production was markedly potentiated by interferon (IFN)‐γ. IL‐1 receptor agonist protein (IRAP) totally blocked NO generation by cytokine‐stimulated astrocytes. Using reverse transcription‐polymerase chain reaction and sequencing analyses of the astrocyte NO synthase gene, we found a single band encoding for a 615 bp product that was identical to the corresponding sequence reported for human hepatocytes. Treatment of human fetal brain cell cultures with IL‐1β plus IFN‐γ resulted in marked neuronal loss, as assessed by microscopic analysis and measurement of lactate dehydrogenase release. This cytokine‐induced neuronal damage was blocked by simultaneous treatment of the brain cell cultures with NMMA or IRAP, suggesting a critical role of IL‐1. These findings indicate that cytokine‐stimulated astrocytes are neurotoxic via a NO‐mediated mechanism and point to potential new therapies for neurodegenerative disorders that involve cytokines and reactive astrocytes. © 1996 Wiley‐Liss, Inc.