During a study of the levels of inhibin and follistatin in ovine amniotic fluid, we noted that although detectable levels of immunoactive inhibin and follistatin were found throughout gestation, the addition of amniotic fluid to a rat anterior pituitary cell culture resulted in a stimulation, rather than the expected suppression, of FSH concentrations. These data suggested the possibility that activin was present in amniotic fluid. We, therefore, set out to isolate the molecules responsible for this activin-like activity and determine their structure. Amniotic fluid, collected from pregnant sheep between 120-140 days gestation, was used as starting material in the purification and diluted in parallel to a human activin-A standard in the activin RIA employed to monitor the purification. A total pool of 7.4 liters amniotic fluid was processed by dye affinity chromatography, hydrophobic interactive chromatography, gel filtration, and a series of reverse phase HPLC steps. Polyacrylamide gel electrophoresis of fractions from the final HPLC step, which showed both activin immunoactivity and bioactivity, revealed a band with a mol wt of 25.3 kilodaltons (kDa), which reduced to 15.8 kDa, and a minor band of 45 kDa, which reduced to 25 kDa. NH2-terminal amino acid sequences of several active fractions from the same region were identical to the known sequence of ovine activin-A. The identification of immunoactive activin, follistatin, and inhibin in amniotic fluid raises the question of the sites of production of these proteins and their interactions and role in fetal physiology.