Three distinct endonucleases (DNase alpha, beta and gamma) were isolated from rat thymocyte nuclei. These three DNases differed in chromatographic behaviors and in apparent molecular masses. The cognate form of acidic DNases alpha and beta did not require divalent cations for their activities, whereas DNase gamma was a neutral endonuclease that required both Ca2+ and Mg2+ for full activity and was inhibited by Zn2+. These enzymes digested HeLa S3 cell nuclear chromatin to the nucleosomal fragments characteristic of apoptosis. Interestingly, apoptotic rat thymocyte nuclei induced by gamma-ray irradiation or glucocorticoid retained considerable activity of DNase gamma, whereas their activities of DNases alpha and beta were markedly reduced. These results indicate that in rat thymocytes DNase gamma is involved in DNA fragmentation during apoptosis.