A new one-step radiolabeled antibody radioassay for measuring free T4 (FT4) in serum (Amerlex-MAB FT4) was evaluated in comparison with an analog tracer RIA of FT4 (Amerlex-M FT4). In this new method, 125I-labeled anti-T4 monoclonal antibody which has cross-reactivity with T3 is used as a tracer. When incubated with serum sample, the tracer binds to FT4 and the remaining tracer binds to a T3 coated particle (Amerlex MAB). The radioactivity bound to Amerlex MAB is measured. Counts of 125I bound to the T3 coated particle were inversely proportional to sample FT4 concentrations. The assay procedure is as follows. Fifty microliter of patient's serum or standard FT4, 500 microliters of Amerlex MAB and tracer is incubated at 37 degrees C for 30 minutes and centrifuged. Then the radioactivity of Amerlex MAB is measured using an autowell gamma counter. The intra-and interassay coefficients of variation were 1.6-2.7% and 2.6-8.0%, respectively. Although Amerlex-M FT4 values were significantly increased by adding human albumin to the serum, Amerlex-MAB FT4 values were not effected by the change of albumin concentrations. In nonthyroidal illness patients, Amerlex-MAB FT4 values were not affected by the concentrations of albumin, TBG and NEFA. The euthyroid central 95% reference range for FT4 determined by Amerlex-MAB FT4 was 0.99 to 1.54 ng/dl. The FT4 levels correlated well with the metabolic status. Although Amerlex-M FT4 values were spuriously increased in patients with anti-T4 autoantibodies, Amerlex-MAB FT4 values were not affected by the autoantibodies. Amerlex-MAB FT4 values of normal pregnant women were slightly lower in the second and third trimesters than in the first trimester. These lower FT4 concentrations in late pregnancy were considered likely not to be artefact by low serum albumin or high serum TBG but to be a physiological event. Amerlex-MAB FT4 values correlated well with FT4 indices and inversely correlated with TSH levels. A significant correlation (n = 401, r = 0.86, p = 0.0001) was observed between Amerlex-MAB FT4 and Amerlex-M FT4 values in various thyroid conditions without antithyroid autoantibodies. In summary, this new assay for FT4 is simple, rapid and reproducible. The measurement is useful for the evaluation of physiological thyroid function and helpful in the management of patients with thyroid diseases.