This noncompetitive, sensitive, immunoradiometric assay of the free alpha subunit of human pituitary glycoprotein hormones is based on two monoclonal antibodies and an avidin-biotin separation system. The affinity of the first antibody, mouse anti-alpha subunit covalently conjugated to biotin, is 3.8 x 10(11) L/mol. The second antibody, radiolabeled with 125I, has an affinity of 5.4 x 10(11) L/mol. A polystyrene ball coated with avidin serves as the separation system. Tests of "purified" immunochemical-grade intact human glycoprotein hormones yielded cross-reactions of approximately 2% in the assay. Sephadex G-100 column chromatography showed that this "cross-reaction" was caused by contamination of the various hormone preparations with free alpha subunit. When the intact glycoprotein hormones were further purified with specific anti-alpha monoclonal antibody, their reaction in the alpha subunit assay was undetectable (less than 0.01%). Interassay CV averaged 3.5%, and intra-assay CV averaged 7.5% at low concentrations of subunit. The detection limit of the assay (0.01 micrograms/L) is adequate to detect free alpha subunit in the blood of normal humans. Mean (SD) concentrations of free alpha subunit in normal humans were as follows: eugonadal men = 437 (35) ng/L; postmenopausal women = 1231 (40) ng/L; eugonadal women, follicular phase = 1061 (40) ng/L; eugonadal luteal phase = 780 (45) ng/L.