A simple and rapid method for the estimation of cellular concentration of thyroxine (T₄), 3,5,3′-triiodothyronine (T₃), 3,3′,5′-triiodothyronine (rT₃), 3,3′-diiodothyronine (3,3′-T₂), and 3′,5′-diiodothyronine (3′,5′-T₂) as well as their distribution between cytosol and membranes in human red blood cells (RBC) is presented. Concentrations of iodothyronines in RBC (RBC-T) were calculated by multiplying the total serum concentrations by the ratio of radioactivity in equal volumes of packed RBCs and serum, pre-incubated with ¹²⁵I-labelled iodothyronines of high specific activity. Plasma and RBC were separated by centrifugation in capillary glass tubes. The separation of membranes and cystosol was performed by hypotone lysis and centrifugation. The median RBC-T of T₄, T₃, rT₃, 3,3′-T₂, and 3′,5′-T₂ from 17 euthyroid subjects were 360 pmol/1, 156 pmol/1, 2.77 pmol/1, 6.81 pmol/1, and 2.17 pmol/1, respectively. The cytosol/cytosol + membrane ration were 66%, 40%, 84%, 77%, and 97%, respectively. The differences in RBC-T were not similar to the differences in free serum concentrations. The ratio of RBC-T to free serum concentration differed considerably between T₄ (16.6), T₃ (24.4), and 3,3′-T₂ (15.5) as compared to rT₃ (5.8) and 3′,5′-T₂ (2.6). Data on three patients with thyroid diseases suggested that RBC-T values were increased in hyperthyroidism and decreased in hypothyroidism, whereas the cytosol/cytosol + membrane-ratio was unaltered.