Two monoclonal antibodies (MK1 and MK2) reacting with human sex-hormone binding globulin (SHBG) were obtained from mice hybridomas. The dissociation constants for the binding of SHBG to MK1 and MK2 were 7.5 and 75 pmol/L, respectively. MK1 was coupled to polyacrylamide beads with a yield of 37%, resulting in 15 mg of antibody per gram of beads. The maximal binding of SHBG by the MK1-beads was 16% of the theoretical capacity. The amount of 125l-labeled MK2 bound to MK1-beads was related to the amount of SHBG present. The system has been used for the immunoradiometric assay (IRMA) of SHBG in serum, and has been standardized with purified SHBG. Assay sensitivity is 3 micrograms/L; intra- and inter-assay (total variation) CVs were 5% and 10%, respectively. Values obtained with the assay for 100 patients' sera agreed well with those obtained with a conventional radioimmunoassay, and SHBG in a patient's serum subjected to gel chromatography eluted as a symmetrical peak with the expected retention when the effluent was analyzed with the present assay. Analytical recovery of SHBG added to serum from a man, a woman, and a pregnant woman ranged between 93% and 107%. The mean (and SD) concentrations of SHBG in sera from healthy women and men were 3.7 +/- 1.1 and 1.8 +/- 0.9 mg/L, respectively.