Using human liver ferritin as a model, we have developed a two-site immunoradiometric assay. Sample (or standard) is incubated with excess rabbit antiserum and 125I-labeled specific sheep antibodies directed against different antigenic determinants until equilibrium is reached. Then excess sheep anti-rabbit immunoglobulin G serum, covalently linked to magnetizable particles, is added. The radioactivity bound to these particles is directly proportional to the amount of ferritin present. The assay is reproducible and precise throughout the clinically important range for ferritin in serum. Late addition of second antibodies coupled to magnetizable particles is a novel, simple, rapid, and universally-applicable separation technique for two-site immunoradiometric assays. By this approach, the first immunological reaction can occur in liquid phase, which shortens the reaction time, maximizes sensitivity, and avoids the need for continuous mixing, as compared with methods in which one of the first antibodies is immobilized on a solid phase.