Immunostainable inhibin alpha-subunit has been demonstrated in rat testes in a pattern consistent with localization in Sertoli cells. In the present study the distribution of alpha-subunit immunostaining was compared to those of beta-A- and beta-B-subunits. Immunostaining of alpha-subunit was present in the seminiferous epithelium of fetal, neonatal, pubertal, and adult rats as well as in Sertoli cells in culture. The distribution of inhibin beta-B-subunit immunostaining in this epithelium was consistent with Sertoli cell localization similar to that of the alpha-subunit. The predominant staining with antibodies against the beta-A-subunit was in nuclei of immature germ cells around the periphery of each seminiferous tubule. The most probable localization of this staining was in the nuclei of pachytene and zygotene spermatocytes. Specific immunostaining with beta-A-subunit antiserum was also evident in the seminiferous epithelium adjacent to the tubular lumen. Immunoreactive alpha- and beta-A-subunit staining was present in a Leydig cell line, and beta-A immunoreactivity was present in interstitial cells of neonatal rat testes. After hypophysectomy, inhibin alpha-subunit immunostaining decreased, beta-A-subunit staining did not change, and beta-B-subunit staining increased. We conclude that immunoreactive inhibin subunits are present in multiple cells in the testis and that the amounts of immunostainable subunits in the seminiferous epithelium are differentially regulated.