Follistatin (FS) is a monomeric protein that binds and regulates the bioavailability of activin. Previously, we found circulating levels of total FS to be similar in men and cycling women. Because relative amounts of activin-bound and free FS are important considerations in determining activin bioavailability, we asked here whether the relative proportions of these two changed during different physiologic states. For this, we developed a two-site, solid-phase, immunochemiluminescent assay for free FS. The assay recognizes the 288 or 315 amino acid variants of human FS and has a detectable limit of 1 ng/mL. Inhibin, transforming growth factor-beta, or alpha-2-macroglobulin do not cross-react or interfere in this assay. Preincubation of FS with activin results in dose-dependent loss of immunoreactivity, confirming specificity of the assay for free FS. Human follicular fluid, pituitary extract, and serum with added FS dilute parallel with the recombinant human FS-288 standard. Recovery of recombinant human FS-288 from serum is quantitative. Using this assay, we found circulating concentrations of free FS to be at or below the detection limit of the assay throughout the menstrual cycle. Comparison of circulating total and free FS levels in postmenopausal or cycling women and normal men suggested that at least 90% is activin-bound. In contrast, measurable quantities of free FS were found in follicular fluid and pituitary extracts. The results of this study, showing that most circulating FS is normally activin-bound, argue against an endocrine role for FS and suggest that a major role of circulating FS is to bind and neutralize the bioactivity of circulating activin. The roles of FS as a local autocrine or paracrine regulator of activin in target tissues, where FS exists in free form, or as an endocrine regulator in human pathophysiology, warrants further investigation.