An immunoradiometric assay (IRMA) for sex hormone binding globulin (SHBG) has been developed in which an 125I-labeled monoclonal antibody [( 125I]S1B5) and a rabbit anti-SHBG antiserum (RAb) are incubated in "liquid-phase" with standards or samples, and RAb-bound complexes are separated using donkey anti-rabbit IgG antibody-coated cellulose. This immunoassay technique is characterized by several advantages; the [125I]S1B5 imparts additional specificity and obviates the requirement for pure SHBG; the use of excess reagents reduces incubation times and also improves assay performance and sensitivity, and incubation in "liquid-phase" conserves and increases the efficiency of the RAb. The assay measures only non-denatured SHBG and is not influenced by the presence of steroid at the binding site. Assay specificity was demonstrated by parallelism between dilutions of pure SHBG and different serum samples. The quantitative recovery of SHBG added to serum, and the agreement between specific activities of SHBG in pure standards and sera, confirm the accuracy of the method. The within and between assay coefficients of variation were less than 7% and less than 11%, respectively, between 12 and 450 nmol/l. The assay sensitivity may be manipulated by altering the concentration of RAb and/or by preincubation with either [125I]S1B5 or RAb, and 0.2 fmol SHBG may be measured on a standard curve. The SHBG assay has been used to measure SHBG concentrations in sera, amniotic fluid, cerebral spinal fluid, seminal plasma and saliva.