Luteal regression is associated with the generation of reactive oxygen species (ROS). To determine the nature of the ROS generator, cells isolated from luteinized rat ovaries were examined for ROS production using luminol-amplified chemiluminescence (LCL). Cells cultured for 2–48 h exhibited minimal LCL, but there was a significant (30- to 50-fold), rapid (maximum at 3–5 min), and dose-dependent increase in LCL in response to phorbol ester (phorbol 12-myristate 13-acetate; TPA; ED50= 0.03 μM) and diacylglycerol (1,2-dioctanoyl-glycerol; ED50= 30 μM). The TPA-induced response was cell number dependent and was virtually abolished by superoxide dismutase, freezing, or heating (95°C for 5 min). Zymosan, known to induce a phagocytic response in leukocytes, stimulated a superoxide (O2−·) response with a slow onset (maximum at 40 to 60 min) and a maximum about one third of that observed for TPA. The response to TPA and zymosan was inhibited by the NADPH/NADH-oxidase inhibitor, diphenylene iodonium (ID50= 5 μM for TPA), but not by the mitochondrial inhibitors, potassium cyanide, rotenone, or sodium azide. Fractionation of cells by centrifugal elutriation showed that TPA-stimulated O2−·production coeluted with the nonsteroidogenic cells and that little, if any, O2−·generation coeluted with the steroidogenic cells. Cells isolated 1, 2, and 4 h after in vivo treatment with a luteolytic dose of prostaglandin F2α(PGF2α) showed a significant increase in TPA-stimulated O2−·production at 2 h, whereas luteal cells or corpora lutea incubated directly with 1 μM PGF2αdid not show any increase in response. Corpora lutea isolated from naturally regressed ovaries (18 days after ovulation) showed a significantly elevated level of TPA-stimulated O2−·production. In conclusion, there is a superoxide generator in luteinized ovaries that is activated through a protein kinase C pathway, localized in nonsteroidogenic cells, transiently increased during PGF2α-induced luteolysis in vivo, and elevated during natural luteal regression.