Dog kidney cytosol contains a high MW (50,000-70,000) and a low MW (approximately 6000) thyronine-binding protein. Low MW cytosol thyronine-binding protein was not previously recognized in cytoplasm. Binding of thyroxine (tetraidothyronine, T4) by the low MW protein had a half-time of association of more than 24 h and accounted for 32% of bound cytoplasmic T4 after 48 h of incubation. Binding of labeled T4 and triiodothyronine [T3] by this moiety was non-dissociable in the presence of 1 .cntdot. 10-5 M unlabeled T4 or T3. The low MW protein existed in a dispersed and apparently aggregated form; the latter eluted in the void volume on Sephadex G-100 and its generation was minimized by 2 mM Ca2+. This binding protein eluted in a fraction which had a high A[absorption]260nm:A280nm ratio, was pentose enriched (orcinol method) and which, because of these characteristics and low susceptibility to digestion by nuclease, was postulated to be a ribosylated cytoplasmic protein or polypeptide. Binding of T4- and T3 by the high MW protein had a half-time of association of 2 h and was saturable. Displacement of labeled T3 from this cytosol thyronine-binding protein was more readily effected with excess unlabeled T4 than with T3, indicating the absence of a T3 specific cytosol thyronine-binding protein site. 3,3'',5''-T3 (reverse T3) was bound with low avidity. Uptake of high MW protein by isolated kidney cell nuclei could not be demonstrated. Binding of T4 by cytosol proteins was independent of pH in the pH range 6.8-8.9, but binding of T3 was minimized at pH 7.4 and enhanced at alkaline pH to the point of equivalency of T4 and T3 binding at pH 8.9. At concentrations of T4 was bound to cytosol thyronine-binding protein, restricting access of this iodothyronine to binding sites in nucleus and mitochondria. Cytosol removed labeled T4 and T3 previously reacted in vitro with isolated cell nuclei; such removal was a linear function of cytosol protein concentration and was blocked by saturation of cytosol thyronine-binding protein with unlabeled iodothyronines. Only the high MW protein accounted for unbinding by cytosol of nuclear hormone.