Ovarian insensitivity to FSH, as observed in some patients suffering premature ovarian failure (POF), could conceivably involve abnormal regulation of local factors that modulate FSH action. Low molecular weight FSH receptor-binding inhibitor (FRBI) has been identified in ovarian follicular fluid and shown to be an antagonist of FSH action. Thus, we undertook these studies to test the hypothesis that elevated FRBI can account for the high serum levels of FSH as measured by RRA relative to RIA values in some POF patients. In order to accomplish this, 2 POF patients were selected from a group of 27 from whom serum FSH had been measured by RIA and RRA. Using a recently developed and validated RRA, FSH was 430 IU (second IRP-78/549)/L and 182 IU/L for serum from patients 1 and 2, respectively. FSH quantitated by RIA was 96 and 136 IU/L in these same serum samples. Thus, the RRA/RIA values for these patients were 4.48 and 1.34. These ratios are: 1) higher than observed for normal cycling women (0.62); 2) higher than observed for normal, postmenopausal women (0.65); and 3) at least 2 SD higher than the mean RRA/RIA ratio of the 27 patients screened. FRBI was separated from FSH in serum from both these patients. FRBI accounted for most of the elevated FSH measured in serum by RRA. The HPLC chromatographic behavior and binding inhibitory activity of FRBI isolated from a large volume of serum from patient 2 were virtually identical to previously observed characteristics of FRBI isolated from porcine follicular fluid. These observations demonstrate that FRBI can account for elevated FSH measured by RRA relative to that measured by RIA. Furthermore, the inhibitor can be biochemically separated from FSH and quantitated by RRA in order to study its postulated relationship to POF. Expanded studies to identify causal relationships between FRBI and ovarian insensitivity to FSH seem warranted at this time.