We measured concentrations of free thyroxin (FT4) in serum by using two new two-step FT4 assays--a solid-phase two-step radioimmunoassay. Spectria, and a time-resolved fluoroimmunoassay. Delfia--and compared the results with those by a two-step FT4 assay (RIA-gnost), a one-step FT4 analog assay (Amerlex-M), and FT4 measured after equilibrium dialysis. The new FT4 assays classified 30 hypothyroid and 43 hyperthyroid patients (untreated) well. In 138 patients with nonthyroidal illness (NTI) and in late pregnancy (n = 36), fewer subnormal FT4 values were reported by Spectria (P less than 0.001), Delfia (P less than 0.001), and RIA-gnost (P less than 0.01) than by Amerlex-M. The results of the Spectria and Delfia methods correlated with the results of the dialysis method (r = 0.76) in NTI patients and pregnancy, and were in better agreement with the clinical state than was FT4 by Amerlex-M. The FT4 values by Amerlex-M, but not by other methods, correlated with albumin concentration. We conclude that these new two-step methods present good alternatives for FT4 analysis.