Follistatin (FST) is a monomeric activin-binding and neutralization protein that has at least three isoforms in human tissues and fluids. The full-length FS315 protein has an acidic 26-residue C-terminal tail that is not present in the shortest form, FS288, due to alternative splicing. An intermediate form, FS303, was identified in follicular fluid that is presumably derived by proteolytic processing of this tail domain. Interestingly, the biochemistry of each of these three isoforms is distinct, including their ability to bind to cell surface proteoglycans, an activity that ranks in the order FS288 > FS303 > FS315. This would suggest that the soluble, circulating FST isoform is likely to be FS315, a hypothesis supported by previous determinations that the serum and follicular fluid forms of FST are biochemically distinct. To test this hypothesis, we developed an immunoassay that is specific for full-length FS315. This assay was validated for use with human serum and follicular fluid samples and then used to examine FST in these fluid compartments. Our results indicate that FS315 is indeed the major circulating FST isoform but is undetectable in follicular fluid samples aspirated from normal women or women with polycystic ovary syndrome. These observations confirm the compartmentalization of FST isoforms according to their biochemical properties and biological actions so that the most soluble form is found in the circulation, whereas the forms that bind to cell surface proteoglycans are found in tissue compartments such as the ovarian follicle. They also confirm that the source of FST in human serum is not the ovarian follicle.