The present study was undertaken and compare the methods for measuring free T3 (FT3) by equilibrium dialysis, free T3 index (FT3I), and T3:T4-binding globulin (T3/TBG) in 40 healthy subjects, 26 patients with hyperthyroidism, 36 patients with hypothyroidism, 16 women in the 3rd trimester of pregnancy, 5 euthyroid subjects with low TBG, and 24 patients with non-thyroidal illnesses (NTI) with normal or low serum T3 concentration. Both FT3I and T3/TBG correlated significantly with FT3 (r = 0.98, P less than 0.001 and r = 0.92, P less than 0.001, respectively) when the data from all subjects were analyzed together. When each group was analyzed separately, FT3I still correlated significantly with FT3 in all groups (r = 0.67-0.97) except in the NTIs with low T3 (r = 0.18). The correlation of T3/TBG with FT3 was also significant in all groups (r = 0.60-0.88) other than the euthyroid with low TBG (r = 0.32) and NTIs with low T3 (r = 0.04). Both FT3I and T3/TBG values agreed well with the FT3 level in hyper- and hypothyroid subjects. In euthyroidism with abnormal TBG level, FT3I agreed well with FT3. However, T3/TBG gave a falsely lower FT3 when the TBG level was high, and a falsely higher FT3 when the TBG was low. In NTIs with normal T3, both FT3I and T3/TBG agreed well with FT3, but FT3I and T3/TBG values were all subnormal in NTIs with low T3, where FT3 ranged from normal to subnormal. These data suggest that 1) both FT3I and T3/TBG may be equally useful for an assessment of FT3 level in hyper- and hypothyroidism, 2) FT3I may be superior to T3/TBG in euthyroidism with abnormal TBG concentration in serum, and 3) neither FT3I nor T3/TBG may be valid for an indirect measure of FT3 in NTIs with low serum concentration of T3.