An ultrafiltration method (UF) for measuring free thyroxine (FT4) and free triiodothyronine (FT3) using the Diaflow YM membrane (Centricon-10) is described. The results are compared with those by equilibrium dialysis (ED) and also by mathematical calculations derived from T4, T3, and binding protein concentrations. The precision with the UF method was excellent. The normal ranges of FT4 and FT3 by the three methods are all comparable. There was a high degree of correlation of FT4 or FT3 results by UF with those by ED and by calculation (r = 0.940-0.974, n = 161, P less than 0.001). FT4 and FT3 by all methods agreed well for hyperthyroidism, hypothyroidism, and for patients with low T4-binding globulin. The mean FT3 in pregnancy was lower than the normal value for all methods, and FT4 concentrations by UF and calculation also decreased in late pregnancy. The mean FT4 by UF and ED in low T3 syndrome were significantly higher than in the normal controls, while the calculated FT4 was lower. The FT3 in low T3 syndrome distributed normal to subnormal in all methods. These results indicate that a) the UF method is a reliable reference method for measuring FT4 and FT3 concentrations; b) the UF results agree well with those by ED and also with theoretically derived values in subjects with thyroid diseases and TBG abnormalities; c) for patients with low T3 syndrome, the FT4 results obtained by UF and ED are similarly discrepant from the calculated results, implying the existence of binding inhibitor(s) which affect both UF and ED measurements.