Two major insulin-like growth factor-binding proteins, IGFBP-2 and IGFBP-3, were identified by Western ligand blotting of human follicular fluid (FF) aspirated from luteinizing follicles during ovarian stimulation with human menopausal gonadotropin (LH and FSH). IGFBP-1 was also present in FF, and an IGFBP with a mol wt of 24000 daltons, present in serum, appeared to be absent in FF. Identification of IGFBP-1, IGFBP-2, and IGFBP-3 was confirmed using IGFBP-specific antisera. IGF-I and IGF-II levels were measured in patient serum and FF at the time of oocyte harvest, and serum levels of both peptides were consistently higher than FF levels. The finding of three major serum IGFBPs in human FF suggests that IGF regulation in the ovary is probably under complex control by this set of regulatory BPs.