Our objective was to identify the endonuclease responsible for DNA degradation in the ovary and determine its localization relative to the developmental state of ovarian follicles. Immature rats were treated with diethylstilbestrol (DES; DES group), DES + eCG (eCG group) or DES + eCG + hCG (hCG group). Nuclei of the eCG and hCG but not the DES group contained a 32/34-kDA DNase I-like endonuclease activity that was Ca2+/Mg(2+)-dependent, stimulated by Mn2+, optimal at pH 7, and identified by anti-DNase I antibody. G-actin, Zn2+, dithiothreitol, aurintricarboxylic acid, and sodium aurothiomalate, but not iodoacetic acid, inhibited the activity. Addition of eCG nuclear protein extracts to nuclei from the DES group induced oligonucleosomal DNA fragmentation, which could be prevented by pretreatment of the extracts with anti-DNase I antibody. DNase I was immunolocalized in nuclei of healthy luteal cells, antral follicle granulosa cells, oocytes of preantral follicles, atretic preantral follicles, and testicular spermatogonia, but was not observed in granulosa cells of preantral follicles, theca cells, antral follicle oocytes, or testicular spermatocytes. Nuclear extracts of rat kidney, liver, and spleen, and bovine, chicken, and human ovaries displayed DNase I-like activity. These results suggest that an endonuclease indistinguishable from DNase I is responsible for ovarian apoptotic DNA degradation.