This report describes the development of a specific and sensitive assay for inhibin B and its application to the measurement of inhibin B concentrations in plasma during the human menstrual cycle. A monoclonal antibody raised against a synthetic peptide from the betaB-subunit was combined with an antibody to an inhibin alpha-subunit sequence in a double antibody enzyme-linked immunosorbent assay format. The validated assay had a limit of detection of 10 pg/mL and 0.5% cross-reactivity with inhibin A. Using this immunoassay, we found that the plasma concentration of inhibin B rose rapidly in the early follicular phase to a peak of 85.2 +/- 9.6 pg/mL on the day after the intercycle FSH rise, then fell progressively during the remainder of the follicular phase. Two days after the midcycle LH peak, there was a short lived peak in the inhibin B concentration (133.6 +/- 31.2 pg/mL), which then fell to a low concentration (<20 pg/mL) for the remainder of the luteal phase. In contrast, the inhibin A concentration was low in the early follicular phase, rose at ovulation, and was maximal during the midluteal phase. The concentration of inhibin B in individual follicular fluid samples was 20- to 200-fold higher than the concentration of inhibin A and was highest in follicular fluid samples from the early follicular phase. Inhibin B appears to be the predominant form of inhibin in the preovulatory follicle. The different patterns of circulating inhibin B and inhibin A concentrations observed during the human menstrual cycle suggest that these forms may have different physiological roles.