The concentrations of inhibin and follistatin in amniotic fluid and in tissue extracts from the placenta, gonads and adrenals of fetal sheep were measured using radioimmunoassays. These tissue extracts were from whole fetuses from days 16 to 45 and from the individual organs from day 46 to 145 (term) and were assayed at multiple dilutions. The capacity of these extracts to alter FSH production of rat anterior pituitary cells in culture was also assessed at multiple dilutions. Immunoactive inhibin concentrations in amniotic fluid from both sexes increased during gestation and levels were significantly greater in males than females. Peak concentrations of immunoreactive inhibin of 11.2 ± 1.9 ng/ml were found in males at 116-125 days of gestation. Follistatin concentrations did not change throughout gestation and no significant difference was noted between sexes. Mean follistatin levels throughout gestation were 3.0 ± 0.9 ng/ml for males and 3.7 ± 0.9 ng/ml for females. Despite the potential for FSH inhibition by inhibin and follistatin, amniotic fluid from both sexes at all stages of gestation stimulated FSH secretion in the pituitary cell bioassays, suggesting the presence of activin which was confirmed by the measurement of immunoactive activin (13.3 ± 2.5 ng/ml) in a specific radioimmunoassay. Maximum concentrations of immunoactive and bioactive inhibin in placental extracts were observed in late gestation (2.2 ± 0.6 and 3.8 ± 1.6 ng/g respectively) and there was no significant difference between sexes. Follistatin concentrations in placental cotyledons ranged from 11.5 to 27.1 ng/g with no significant difference between sexes. In view of the higher follistatin concentrations compared with inhibin, it is likely that the capacity of placental extracts to suppress FSH production by pituitary cells in culture is due predominantly to follistatin. Immunoactive inhibin was observed in high concentrations in the fetal testis throughout gestation; with concentrations increasing to a maximum of 1993.0 ± 519.7 ng/g at 126- 135 days of gestation with a ratio of bioactive: immunoactive inhibin of 1:20. Although bioactive and immunoactive inhibin was also observed in fetal ovaries and adrenals from both male and female fetuses, concentrations were lower than those observed in fetal testes. Follistatin concentrations in the fetal testis were elevated between 70 and 95 days (97.6 ng/g) and then declined. Similar concentrations were found in the adrenal glands of both sexes (males 83.5-103.3 ng/g: females 55.3-95.8 ng/g). In both males and females, immunoactive inhibin concentrations in fetal adrenals increased during gestation peaking at levels of 34.4 ± 16.5 and 27.8 ± 9.0 ng/g respectively. These data suggest that the capacity of adrenal extracts to suppress FSH production by pituitary cells is due to both inhibin and follistatin. These studies demonstrated that significant concentrations of immunoactive inhibin and follistatin are present in amniotic fluid, and the fetal gonads, adrenal glands and placenta in sheep. The role of these proteins during fetal development requires further study.