Two forms of purified follistatin, a single-chain polypeptide of mol wt 35,000 (35 Kd) protein, and a related molecule of mol wt 32,000 (32 Kd), which differs from the 35 Kd form in glycosylation or carboxyl terminal truncation, specifically inhibit the release of immunoreactive FSH by primary cultures of rat pituitary cells. Both forms of follistatin and inhibin-A give similar dose-response curves, with identical slopes and maximal effects, suggesting that they may all act through the same mechanism on the pituitary cells. The median effective dose (ED50) of each of the follistatins is 6.2-7.3 ng/ml (1.8 x 10(-10) M), which corresponds to approximately 1/3 of the potency of inhibin. The effect of 35 Kd or 32 Kd follistatin is highly specific for suppressing the release of immunoreactive FSH since there is no demonstrable concomitant effect on the secretion of other pituitary hormones. The effect of follistatins, like that of inhibins, is different from that of the hypothalamic hypophysiotropic factors, requiring greater than or equal to 18 h of incubation in a pituitary monolayer culture system to demonstrate. Coincubation of inhibin and follistatin shows an additive effect in the suppression of FSH release. Pituitary cells exposed to follistatin have significantly less depletion of intracellular FSH (0.01) than those treated with inhibin, indicating that follistatin may act primarily on the suppression of FSH release rather than on both release and synthesis of FSH, as is the case with inhibin.