Proliferation of Sertoli cells in the rat testis occurs only during the perinatal period and is maximal during fetal life. This interval is thus of critical importance in establishing the complement of Sertoli cells that populates the adult testis. FSH has been implicated in this process, but direct evidence in support of its involvement is lacking. In the present study, we have used in vivo and in vitro approaches to determine whether FSH produced by the fetal pituitary has a role in regulating Sertoli cell division in the fetal testis of the rat. On day 18 of gestation, just before the onset of maximal Sertoli cell proliferation, fetuses were either decapitated in utero or given antiserum to FSH. Light microscope autoradiography was then used to compare uptake of [3H]thymidine by Sertoli cell nuclei in testes from decapitated or antiserum-treated fetuses to that in corresponding controls on the following day. Both treatments produced dramatic and equal reductions in the percentages of Sertoli cells preparing to divide on day 19, suggesting that FSH from the fetal pituitary stimulates Sertoli cell proliferation in fetal testes. The effect of FSH or (Bu)2cAMP on Sertoli cell proliferation was also studied in vitro by placing testes from intact or decapitated fetuses into organ culture, with or without exogenous hormone or cyclic nucleotide. In all cases, [3H]thymidine was present for the final 4 h of culture. When testes were placed into medium containing isotope immediately after their removal from the fetus, the difference in labeling between testes from intact and decapitated fetuses was similar to that measured in vivo. After testes from decapitated fetuses were cultured for 8 h with or without FSH or (Bu)2cAMP, labeling of Sertoli cells in the treated group increased markedly over that in untreated cultures. After 28 h of exposure to FSH or (Bu)2cAMP, labeling in testes from decapitated fetuses remained significantly higher than that in corresponding untreated controls. In contrast, when testes from intact rats were cultured for 8 h in the presence of either cAMP or FSH, (Bu)2cAMP, but not FSH, brought about an increase in the percentage of Sertoli cells labeled compared to the control value. However, after exposing these testes to either FSH or (Bu)2cAMP for 28 h, the percentage of Sertoli cells labeled was greatly enhanced. Taken together, the data obtained from these experiments identify FSH as a major factor in controlling expansion of the Sertoli cell population during fetal development of the rat.(ABSTRACT TRUNCATED AT 400 WORDS)