A binding protein for activin was purified from bovine pituitary by affinity chromatography on dextran sulfate-Sepharose CL-4B and activin-Affi-Gel 10. A 52,700-fold purification over the starting crude homogenate was achieved. The purified preparation showed two bands of 36 and 33 kilodalton in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. The ability of each form of the protein to specifically bind activin was determined by ligand blot analysis and binding competition study. Each protein was found to have the same NH2-terminus and its sequence was identical to that of follistatin, which is a specific inhibitor of identical to that of follistatin, which is a specific inhibitor of FSH release. Moreover, the binding protein was shown to inhibit the spontaneous FSH release from cultured pituitary cells as does follistatin. These properties are the same as activin-binding protein that we have obtained from rat ovary. These results support a conclusion that activin-binding protein/follistatin exists also in the pituitary. Activin-binding protein has an ability to inhibit the activin-induced augmentation of FSH release from cultured pituitary cells as does inhibin. However, the inhibitory pattern by the binding protein was quite distinct from that of inhibin, suggesting that there may be different mechanism(s) for their antagonistic actions. Stoichiometric inhibition as shown by gel filtration analysis indicates that activin-binding protein binds activin to form an inactive equimolar complex having neither stimulatory nor inhibitory activity for FSH secretion by the pituitary. These findings suggest that activin is actually involved in FSH secretion in the pituitary and that the activin action in the pituitary is regulated by activin-binding protein/follistatin.