A radioimmunoassay for inhibin in bovine and human follicular fluid (bFF, hFF) and serum from both species was developed based on an antiserum raised in a rabbit to purified bovine 58 kDa inhibin. Following immunization, parallel changes in plasma FSH and inhibin antibody titre were observed suggesting inhibin neutralization in vivo. The antiserum neutralized bFF, hFF and purified 31 kDa and 58 kDa inhibin activity in an in vitro bioassay system. Purified 58 kDa and 31 kDa inhibin were iodinated using a chloramine-T procedure and the 125I-inhibin purified by elution from Matrex Red A, with the iodinated molecules showing similar physicochemical properties to non-iodinated inhibin. Both tracers were stable in bFF but only 125I-31 kDa inhibin was stable in serum. A second antibody RIA system using either tracer yielded a parallel displacement between purified 31 kDa and 58 kDa inhibin. The cross-reaction in the RIA of inhibin from different species when expressed in terms of their bioactivity was bFF 100%, hFF 30%, ovine FF less than 1% and rat ovarian extract non-detectable. Rat LH and FSH, ovine LH and FSH, hCG, bovine TSH, LHRH, ovalbumin and bovine serum albumin showed less than 0.5% cross-reactivity using either tracer. Similar profiles of both bio- and immunoactive inhibin were observed at each stage of the inhibin purification procedure. The in vitro biological to immunological ratios for a number of purified 31 kDa and 58 kDa inhibin preparations using both tracers in the RIA, ranged from 0.30 to 0.43. The RIA was modified for serum by using 125I-31 kDa inhibin as tracer and an elevated temperature (30 degrees C) to minimize non-specific effects. No detectable activity was determined in steer or human post-menopausal serum whilst bull and human female serum showed parallel dose-response curves to their respective follicular fluid standards with circulating levels of 0.9 and 1.1 ng/ml respectively.