We describe a double-antibody radioimmunoassay for the determination of free thyroxine (FT4) in serum using a 125I-labeled T4-IgG conjugate of very large molecular weight (approximately 1,000,000) as tracer. The binding of this conjugate to the anti-T4 antibody was found to be satisfactory and independent of the T4-binding protein concentration. The T4 sequestration by the antibody was limited and the sample dilution used in the final assay mixture was low. The results determined by the assay developed for samples from hypo-, hyper-, euthyroid subjects and from non-thyroidal-illness patients were well correlated with those obtained by a free T4-equilibrium dialysis kit (Nichols). The detection limit of the assay was 0.41 pmol/l and the range covered by assay standard solutions was 2.4-121 pmol/l. The FT4 concentrations in serum as determined by this assay were 9.6-31 pmol/l (mean 21 pmol/l) for 46 euthyroid serum samples; 6.7-47 pmol/l (mean 23.5 pmol/l) for 27 non-thyroidal illness; and 4.2-25.8 pmol/l (mean 11.6 pmol/l) for 19 pregnant women.