Triiodothyronine (T3) may bind directly to receptors present in liver cell nuclei, or may be transported into nuclei by receptor protein(s) present in the cytosol. To evaluate these possibilities, T3 binding was studied in vitro using liver cell nuclei isolated from rats exposed in vivo to very low (H), normal (N),or high levels of T3 (H + T3), and using nuclei incubated in vitro with added cytosol proteins. Ka for T3 was 0.075 +/- 0.05 x 10(10) M-1 in N, 0.1 + 0.04 in H, and 0.094 + 0.04 in H + T3, and pg T3 bound/100 mug DNA were 47 +/- 17, 31 +/- 14, and 29 +/- 8 in the three groups. The data indicate no difference in binding capacity between the groups related to prior in vivo exposure to T3, and that T3 may bind directly to empty nuclear receptor sites. Rat liver cytosol proteins added to the in vitro incubation medium always depressed T3 uptake by nuclei. Bovine serum albumin had a similar effect. Large amounts of rat serum proteins depressed uptake, but low levels augmented T3 binding through an unknown mechanism. It is probable that free T3 in serum is in equilibrium with free T3 in the cytosol and nucleus, and binds directly to nuclear receptor proteins without mediation by a cytosol receptor protein.