Follistatin is a monomeric protein first identified in and isolated from ovarian follicular fluid. Evidence that follistatin might be an ovarian endocrine hormone functioning in a negative feedback fashion to modulate pituitary FSH production is based primarily on in vitro experiments. To examine the possible role of follistatin as an endocrine agent in vivo, we sought to relate circulating levels of follistatin to ovarian activity in women. Therefore, we developed a specific and sensitive homologous RIA using antiserum generated against recombinant human follistatin for the measurement of total follistatin in the presence or absence of activin. Follistatin was measured quantitatively (106 +/- 6% recovery) using calibration standards ranging from 0.4-25 ng/tube and up to 400 microL/tube serum. Furthermore, all of the endogenous follistatin measured in human serum could be removed by adsorption to activin-coated plates. Using this homologous RIA, human follicular fluid (100-600 ng/mL; n = 75) contained 3-150 times more follistatin than serum (4-35 ng/mL), an observation consistent with the notion that serum follistatin originates from the gonad. However, further studies of follistatin levels across the normal menstrual cycle (mean +/- SE, 8.09 +/- 0.73; n = 72 daily samples from 4 women), in pregnant women (17.49 +/- 1.34; n = 8), in daily samples from 20 women undergoing ovarian stimulation by exogenous FSH (9.90 +/- 0.62; n = 119), in postmenopausal women including two ovariectomized individuals (9.57 +/- 0.43; n = 8), and in GnRH-deficient women (9.85 +/- 0.50; n = 6) failed to support the hypothesis that serum levels of follistatin reflect ovarian activity in women. Levels of follistatin measured in serum collected across normal menstrual cycles did not fluctuate. However, the roughly nanomolar concentrations of follistatin measured suggest a physiological role for this protein. Follistatin at nanomolar concentrations may be capable of binding and inactivating circulating activin and perhaps in this way limiting the biological activity of activin to local autocrine or paracrine mechanisms. Measurement of peripheral levels of follistatin apparently represents only a first, albeit crucial, step in the study of the physiological significance of this protein in human reproduction.