Many studies demonstrated the presence of cytosolic thyroid hormone binding proteins (CTHBPs) in various tissues, but the physiologic significance of these CTHBPs is not clear, partially because of the lack of information about the physicochemical properties of CTHBPs as purified forms. Since the difficulty in isolating these CTHBPs is considered to be due to instability during the various procedures for their isolation, studies on the stability of CTHBPs of rat liver were performed using a charcoal binding method to separate bound and free hormones. Binding characteristics of CTHBPs of rat liver were also determined. Specific triiodothyronine (T3) binding sites of cytosolic T3 binding protein (CT3BP) of rat liver were destroyed as the time progressed in homogenate at 0 degrees C, and Aprotinin (500 U/ml) had little effect in protecting these binding sites. T3 binding sites were stable in the form of cytosol at -20 degrees C up to 10 weeks. Dithiothreitol (DTT) had no effect on T3 binding to cytosol. T3 binding to CT3BP was pH-dependent with maximum specific binding at pH 7.4. T3 binding to CT3BP was stable at 4 degrees C overnight but was destroyed rapidly at 37 degrees C. Interestingly, specific T3 binding sites of CT3BP were completely abolished by dialysis, and Ca2+ or Mg2+ had no effect on retaining the specific binding sites. Thus, CT3BP was supposed to require some dialysable small molecule(s) to maintain specific T3 binding sites. Scatchard plot of T3 binding to crude cytosol revealed a high affinity, limited capacity T3 binding site with affinity constant (Ka) of 5.9 X 10(7) M-1 and maximum binding capacity (MBC) of 118 ng/g. liver. Relative affinities of T3 analogues for CT3BP were determined by comparing the molar concentrations of T3 analogues required for 50% inhibition of tracer 125I-T3 binding. If the affinity of L-T3 was assigned 100, D-T3 would have a value of 66.1; L-T4, 22.3; D-T4, 16.5; Triac, 6.2; and both Tetrac and reverse T3 were less than 1. Thus, the binding characteristics of CT3BP were fundamentally different from those of nuclear T3 receptor. Cytosolic thyroxine (T4) binding protein (CT4BP) of rat liver was relatively stable compared with CT3BP in homogenate at 0 degrees C. CT4BP was also stable in the form of cytosol at -20 degrees C for 10 weeks. CT4BP was pH-dependent with maximum specific binding at pH 7.4. It was stable at 4 degrees C overnight but destroyed rapidly at 37 degrees C. Specific T4 binding was decreased by dialysis but not abolished completely.(ABSTRACT TRUNCATED AT 400 WORDS)