Methods for separating bound and free fractions in steroid enzyme immunoassay with a monoclonal antibody are described. Three liquid-phase separation techniques and related preincubation methods were studied using β-galactosidase and horseradish peroxidase as labels. In the double- and triple-antibody systems, the effects of concentrations of first, second and third antibodies, including anti-immunoglobulin G Fc fragment antisera, and incubation times on immune precipitation were examined. Differences between the two enzyme labels in immunoreactivity were observed in some cases; this can be explained in terms of a steric interaction. Under optimal conditions, a dose-response curve with a high sensitivity was obtained in each 11-deoxycortisol assay system.