We previously reported that incubation of cultured astrocytes in Ca² + ‐containing medium after exposure to Ca² + ‐free medium caused Ca² +  influx followed by delayed cell death. Here, we studied the mechanisms underlying the Ca² + ‐mediated injury of cultured astrocytes. Our results show that Ca² +  reperfusion injury of astrocytes appears to be mediated by apoptosis, as demonstrated by DNA fragmentation and prevention of death by caspase‐3 inhibitors. Paradoxical Ca² +  challenge stimulated rapidly reactive oxygen species (ROS) production. Ca² +  reperfusion injury of astrocytes was influenced by several reagents which modified ROS production. When astrocytes were exposed to hydrogen peroxide (H₂O₂) for 30 min and then incubated without H₂O₂ for 1–5 days, cell toxicity including apoptosis was observed. Ca² +  reperfusion injury induced by Ca² +  depletion or H₂O₂ exposure was blocked by the iron chelator 1,10‐phenanthroline, the NF‐κB inhibitor pyrrolidinedithiocarbamate and the calcineurin inhibitor FK506. Incubation in normal medium after H₂O₂ exposure rapidly increased the level of nuclear NF‐κB p65 subunit, and the effect was blocked by 1,10‐phenanthroline, pyrrolidinedithiocarbamate and FK506. These findings indicate that Ca² +  reperfusion‐induced apoptosis is mediated at least partly by ROS production and ROS cause NF‐κB activation in cultured astrocytes.