Prostaglandins may play an important role in regulating normal renewal of gastrointestinal epithelium, epithelial injury repair, and initiation or progression of intestinal neoplasia. Synthesis of prostaglandins is catalyzed by either of two cyclooxygenase isoforms, Cox-1 and Cox-2. Cox-1 is the predominant cyclooxygenase isoform found in the normal intestine. In contrast, Cox-2 is present at low levels in normal intestine but is elevated at sites of inflammation and in adenomas and carcinomas. To determine directly whether prostaglandins synthesized by Cox-1 or Cox-2 regulate crypt epithelial cell fate after genotoxic or cytotoxic injury, we examined apoptosis, prostaglandin synthesis, and crypt stem cell survival after γ-irradiation in Cox-1^−/− and Cox-2^−/− mice. Cox-1^−/− mice had increased crypt epithelial cell apoptosis and decreased clonogenic stem cell survival compared with wild-type littermates. PGE₂synthesis was also diminished in Cox-1^−/− mice compared with wild-type controls in unstressed intestine and after radiation injury. In contrast, apoptosis, stem cell survival, and intestinal PGE₂ synthesis in Cox-2^−/− mice after irradiation were the same as in wild-type littermates. Crypt stem cell survival after irradiation was inhibited by a highly specific neutralizing antibody to PGE₂, suggesting that this prostaglandin mediates stem cell fate in vivo. These data suggest that prostaglandins synthesized by Cox-1 regulate multiple steps that determine the fate of crypt epithelial cell after genotoxic or cytotoxic injury.