Serum binding proteins (BPs) have been identified for several peptide and protein hormones, and their presence has significant implications for the biological action of the hormone. Follistatin (FS) has been identified as an activin- and inhibin-BP in tissues, serum, and follicular fluid of several species, including humans. In this study, the binding kinetics of FS for activin and inhibin were characterized in human serum using gel filtration chromatography and compared to those of pure recombinant hormones using chromatography and a new solid phase assay. When complexed with radiolabeled activin or inhibin, FS eluted at a volume corresponding to a mol wt range of 67,000-150,000, an elution volume identical to the lower mol wt BP peak observed in serum. Furthermore, kinetic analyses of recombinant FS binding to activin using a solid phase assay revealed that 1) the FS-activin interaction is of high affinity, similar to or exceeding that estimated for activin binding to its receptor; 2) binding to activin is essentially irreversible at physiological pH; and 3) the potency of inhibin is approximately 500- to 1000-fold lower than that of activin in the FS binding assay. The lack of FS-[125I]activin complex reversibility observed in the solid phase assay was confirmed using a modified gel filtration chromatography protocol. Thus, preincubation of pure FS or serum with unlabeled activin for 2 h eliminated all binding of subsequently added labeled activin despite a much longer incubation period. However, when labeled activin was incubated with FS for 2 h, subsequent addition of unlabeled activin or inhibin was unable to displace labeled activin from FS, again demonstrating a lack of reversibility. Finally, to map this high affinity interaction, overlapping synthetic peptides were used to compete with labeled activin for FS binding. Two potential contact sites between FS and activin were identified, one near the N-terminus (amino acids 15-29) and the other near the C-terminus (amino acids 99-116). Given its apparently irreversible nature, high affinity, and ability to neutralize activin's biological activity, FS is quite different from the typical hormone-BP. These unique properties of FS undoubtedly attest to the potency of activin in many physiological and developmental settings and, therefore, to the importance of BPs, such as FS for regulating activin's bioactivity, distribution, and/or clearance.