Mullerian Inhibiting Substance (MIS) production in rat testes from the late fetal to the adult period and its modulation by gonadotropins in neonatal testes were studied using immunohistochemistry, northern analysis, and a graded organ culture bioassay for MIS. The intense immunohistochemical staining for MIS seen in fetal and newborn testes began to decrease gradually after the third postnatal day, then decreased dramatically on the fifth postnatal day. MIS immunohistochemical activity was then present at a low level until about the 20th postnatal day, after which it was barely detectable. The testes from rats treated with FSH at birth showed a considerable drop in MIS immunohistochemical activity on the third postnatal day to 29% of control testes, and a less profound decrease on the second and fourth postnatal days to 46% and 61% of control, respectively; thereafter MIS levels were the same in treated and untreated animals. With shorter courses of FSH treatment, immunohistochemical staining showed less depression of MIS on the third day, and no difference by the fourth postnatal day, indicating that the inhibitory effect on testicular MIS production may require continued FSH exposure. Three-day testes that had been treated with FSH for 2-1/2 days had less MIS messenger RNA compared to control testes of the same age, suggesting that the inhibitory effect of FSH on MIS production could be transcriptionally mediated. In contrast LH treatment produced no difference in either messenger RNA expression or immunohistochemical staining for MIS. These findings suggested that FSH may be a modulator of MIS production in neonatal testes.