Antioxidant defenses play a critical role in the regulation of programmed cell death, even when death is induced by nonoxidative stimuli. During spermatogenesis, most of the testicular germ cells degenerate by an apoptotic process that is under hormonal control. However, the exact mechanisms by which hormonal signals are transduced within the cells to direct their life, and whether other effectors of the apoptotic pathway, for example antioxidants, take part in the control of human germ cell survival, are not known. In the present study, testosterone and N-acetyl-L-cysteine (NAC), which is an antioxidant, an inhibitor of apoptosis in several systems, and a survival factor in human semen, were found to suppress programmed cell death in human testicular germ cells in vitro. The samples came from adult men undergoing orchidectomy for prostate cancer. Germ cell death was induced by incubating segments of seminiferous tubules under serum-free culture conditions. This apoptosis, detected by Southern blot analysis of DNA fragmentation, by DNA labeling in situ, and by morphological analysis under the electron microscope, was significantly inhibited by testosterone at concentrations of 10(-6) and 10(-7) mol/L. NAC concentrations of 125, 100, 50, and 25 mmol/L suppressed germ cell death in a dose-dependent manner. This inhibition was effective during 4, 24, and 48 h of incubation. Apoptotic cells were identified mainly as spermatocytes and early spermatids. Programmed cell death was also demonstrated in late spermatids. We conclude that NAC, which is an antioxidant, plays an important role in germ cell survival in the human seminiferous tubules in vitro. We also suggest NAC as a possible new therapeutic factor for some men with idiopathic oligospermia.