The enzyme glutathione peroxidase 1 (GPx1) is involved in the cellular detoxification of peroxides. To test for the consequences of GPx deficiency in astrocytes, astrocyte-rich primary cultures from wild-type and GPx1-deficient [GPx1(–/–)] mice were exposed to H2O2. In GPx1(–/–) astrocytes, the clearance rate of H2O2 was slower than in wild-type cells. In contrast to GPx1-deficient astrocytes, wild-type cells exhibited, within 2 min of H2O2 application, a rapid and transient accumulation of cellular glutathione disulfide that amounted to 60% of total glutathione. The peroxide treatment did not affect the viability of wild-type astrocytes, whereas 45% of the GPx1(–/–) cells died within 8 hr. However, the viability of both types of astrocytes was strongly compromised by lowering cellular glutathione content before peroxide application. In contrast, inactivation of catalase caused substantial cell death only in GPx1(–/–) cells but not in wild-type astrocytes. The cell death observed was prevented by the iron chelators deferoxamine, 1,10-phenathroline, or 2,2′-dipyridyl, whereas preincubation with ferric ammonium citrate increased the toxicity of peroxide treatments. These results demonstrate that GPx1 contributes to the rapid clearance of H2O2 by mouse astrocytes and that both GPx1 and a high concentration of glutathione are required to protect these cells from iron-dependent peroxide damage. © 2006 Wiley-Liss, Inc.