We have devised a practical, sensitive and specific method for simultaneous measurement of free thyroxine (FT₄) and free triiodothyronine (FT₃) in undiluted serum by direct equilibrium dialysis radioimmunoassay (RIA). Two hundred microliters serum sample was dialyzed against buffer (pH 7.4) for 20 hours at 37°C and ∼800 μL of the dialysate was used for measuring FT₄ and FT₃ simultaneously. The assay was set up in polystyrene tubes coated with anti-T₄ antibody and available commercially for FT₄ measurement (Quest-Nichols Institute, San Juan Capistrano, CA). The mean ± SE (range) FT₄ concentration (ng/dL) was 1.2 ± 0.04 (0.7.0 to 2.30) in 54 normal subjects. It was significantly increased (3.6 ± 0.4 [1.8 to 9.6], n = 20) in hyperthyroidism and clearly decreased (0.40 ± 0.04 [1.10 to 0.70], n = 26] in hypothyroidism. All nonthyroid illness (NTI) patients had normal FT4 except 3, 2 of whom were on amiodarone and 1 had received heparin. Serum FT₄ concentration was minimally elevated in 18 newborn cord blood serum (1.40 ± 0.08 [0.90 to 2.2], cf. normal p <.05). The mean serum FT₃ concentration (pg/dL) was 285 ± 10 (134 to 454) in 54 normal sera. It was clearly increased in hyperthyroidism (1033 ± 98 [593 to 2134], n = 20, p <.001). However, serum FT3 varied widely in hypothyroidism (27 to 597, mean 235 ± 24, NS) as did serum total T₃ (19 to 175). Interestingly, however, the mean serum FT₃ concentration was normal (273 ± 28 [62 to 575, NS]) in 25 NTI patients. All of these patients had low serum total T₃ (46 ± 5.0 [10 to 84], ng/dL; normal 84 to 160, p < 0.001), while FT₃ was clearly normal in 21 of 25 patients and low in the remaining 4 patients. Similarly, among 18 newborn cord blood sera serum FT₃ concentration was normal in 15 and subnormal only in the remaining 3 while all had clearly subnormal total T₃ (28 to 74 ng/dL). Conclusions: (1) A practical, sensitive, and specific assay for simultaneous measurement of FT₄ and FT₃ is described; (2) FT₃ is consistently elevated in hyperthyroidism while FT₄ is elevated in most (∼85%) cases; (3) FT₄ is consistently decreased in hypothyroidism but FT₃ varies widely; (4). Serum FT₃ concentration is normal in ∼83% of patients with the low T₃ syndrome in NTI and newborn cord blood serum. These data suggest that normal FT₃ may explain clinical euthyroidism in many patients with the low T₃ syndrome.