In previous studies, cyclooxygenase (COX)-1 and COX-2 isozymes have been detected in the rat epididymis. COX-1 mediates electrolyte and fluid secretion induced by a number of peptide hormones, including bradykinin, angiotensin, and endothelin, via local formation of prostaglandin (PG) E₂; however, the physiological role of COX-2 remains largely unknown. Marked apoptotic cell death in the rat epididymis following androgen depletion has been reported. Because expression of both COX isozymes is dependent on androgen, we investigated whether these isozymes control apoptosis in the epididymis. Apoptosis was detected in rat epididymal epithelial cells by in situ staining using the TUNEL method and by the presence of internucleosomal DNA fragmentation using capillary electrophoresis with laser-induced fluorescence detection. Specific COX inhibitors were used to delineate the roles of the 2 isozymes. There was no significant apoptotic cell death in normal and specific COX-1 inhibitor (SC-560)-treated epididymal cells. However, application of a specific COX-2 inhibitor (NS-398) induced apoptosis in a dose- and time-dependent manner. A similar apoptotic effect of COX-2 inhibitor was seen in the in vivo study. The drastic DNA fragmentation induced by COX-2 inhibitor could be reversed completely by PGD₂ and partially by PGE₂. In addition, the protective effect of PGD₂ against COX-2 inhibition was significantly blocked by a PGDP-receptor antagonist, BWA868C. These results indicate that the COX-2 products PGD₂ and, to a lesser extent, PGE₂ control apoptosis in cultured rat epididymal cells in vitro.