A reliable method has been developed for the determination of total serum T3, dialyzable fraction (DFT3), and absolute concentration of free T3 (AFT3). Total T3 values (mean +/- SD) were: healthy euthyroid subjects, 0.33 +/- 0.07 mug per 100 ml; hyperthyroid patients, 0.71 +/- 0.1 mug per 100 ml; hypothyroid, 0.10 +/- 0.03 mug per 100 ml. Values (mean +/- SD) for DFT3 in these groups were 0.46 +/- 0.14%, 0.78 +/- 0.17%, and 0.16 +/- 0.08%, respectively. Calculated values for AFT3 were: 1.51 +/- 0.4 mmug per 100 ml, 5.00 +/- 0.6 mmug per 100 ml and 0.24 +/- 0.1 mmug per 100 ml, respectively. Dilution of serum before dialysis lowered estimated DFT3 values. Enrichment of serum with labeled T3 in the range examined did not affect DFT3. However, DFT3 was increased by addition of Merthiolate to serum in concentration 1: 10,000 due to displacement of T3 from thyroxine-binding globulin to albumin. The data suggest that triiodothyronine may play a considerably more important role in normal and pathological physiology, as evidenced by kinetic analysis using these data. A metabolic role for T3 equal to that of T4 is indicated.