Recent investigations in our laboratory revealed divalent cation-dependent endonuclease activity in testes from 2-methoxyethanol-treated rats, which was able to cleave substrate DNA into a pattern of DNA fragmentation consisting of approximately 180-200 base pairs. Further studies were undertaken to characterize the active nuclease. F344 rats were treated with 2-methoxyethanol, a glycol ether that causes the death of pachytene spermatocytes in juvenile and adult rats. The active nuclease was found in nuclear extract from treated animals, but not controls. A radioactive gel nuclease assay, which detects degradation and loss of 32P-labeled DNA from a DNA-containing polyacrylamide gel, localized the nuclease activity to a band of approximately 18 kDa. This activity was dependent on calcium and was inhibited by both zinc and aurintricarboxylic acid. Amino acid sequence data showed that this protein was identical to cyclophilin A. Immunohistochemistry using antibodies against cyclophilin A found specific staining in pachytene spermatocytes, spermatids, interstitial cells, and Sertoli cell nuclei. Cyclophilin A staining was present in both control and 2-methoxyethanol-treated rat testes in a stage-dependent manner, with pachytene spermatocytes in stage-VIII-XIV seminiferous tubules most heavily stained. These data demonstrate that rat testis germ cells contain relatively high levels of cyclophilin A whose nuclease activity is associated with spermatocyte apoptosis induced by 2-methoxyethanol.