Inhibins, activins, and follistatins are all believed to play roles in the regulation of FSH secretion by the pituitary and in the paracrine regulation of testis function. Previous studies have resulted in conflicting data on the pattern of expression of the inhibin/activin subunits, and little information on expression of follistatin during fetal/neonatal life. We have made use of new, highly specific monoclonal antibodies and fixed tissue sections from fetal, neonatal, and adult rats, and limited amounts of fetal and neonatal human testis, to undertake a detailed immunocytochemical study of the pattern of expression of these regulatory proteins. In the rat, positive immunostaining for the alpha-subunit of inhibin (alpha) was first detectable on day 14.5 post coitum (p.c.), the first day on which the testis could be morphologically distinguished from the ovary. During fetal life, the alpha-immunostaining was most prominent in the fetal Leydig cells. In Sertoli cells, alpha-immunostaining was slightly stronger on days 14.5 and 15.5 p.c. compared with 16.5-20.5. After birth, alpha-immunostaining remained intense in fetal Leydig cells but declined following their replacement with their adult-type counterparts; in contrast, alpha-subunit increased in Sertoli cells immediately after birth. Immunostaining with antibodies specific to betaB-subunit showed a similar pattern to that of the alpha-subunit, except that positive immunostaining was first detectable on day 16.5 p.c., 2 days later than immunostaining for the alpha-subunit. The pattern of betaB-immunostaining in postnatal samples paralleled that of the alpha-subunit. Immunostaining using antibodies against the betaA-subunit did not produce any significant reaction product in any sample. Follistatin was undetectable in the fetal rat testis but appeared in the Leydig cells immediately after birth and its expression remained intense throughout postnatal development and in adult testis. No evidence was obtained for expression of either the inhibin/activin subunits or follistatin in the germ cells, peritubular myoid cells, or other interstitial cells in any of the sections examined. In the human fetal testis, both alpha- and betaB-subunits were immunodetectable at 16, 18, and 24 weeks gestation in Sertoli and Leydig cells, with stronger immunostaining in Sertoli cells at 24 weeks. Postnatally at 4 months, immunoexpression of the betaB-subunit was no longer detectable, whereas the alpha-immunostaining became weaker but was still present in both Sertoli and Leydig cells. No positive immunostaining for betaA-subunit or follistatin was detectable at any time point studied. In conclusion, we have shown that, in the rat testis, the majority of inhibin alpha-subunit and inhibin/activin betaB-subunit is immunolocalized to the fetal-type Leydig cells during fetal/neonatal life but, following birth, immunoexpression in the Sertoli cells of both subunits increases markedly while follistatin is immunodetectable only postnatally.