125I-Triac was employed to measure hepatic thyroid hormone nuclear receptor (RT) in the rat. The binding properties of 125I-Triac and 125-T3 were compared in a 0.4 M KCl extract of a liver nuclear preparation. The order in which the stable compounds, Triac, T3, T4and rT3, competed for125I-Triac and125I-T3binding in liver nuclear extract was similar (Triac>T3>T4>rT3), suggesting association of both radioligands with RT. Scatchard plot analysis of specific125I-Triac and125I-T3binding in nuclear extract gave approximately equal estimates of the maximum binding capacity (MBC). However, the binding affinity, as represented by the equilibrium association constant (KA), was higher for125I-Triac than for125I-T3 (7-10×109M−1vs 1-3×109M−1). To determine the effect of contaminating serum proteins on estimates of MBC and KA, a small amount of dilute rat serum was added to the same nuclear extract preparation. Addition of serum decreased the KAvalue and markedly increased the MBC values estimated by analysis of125I-T3binding data. In contrast, KAand MBC values derived from125I-Triac binding data were not influenced appreciably by the addition of serum. These data indicate that: 1) both125I-Triac and125I-T3bound to RT in rat liver nuclear extract, 2) the affinity of RT for125I-Triac is appreciably greater than for125I-T3, and 3) estimates of RT concentration (MBC) made with125I-Triac are less sensitive to serum protein contamination than those made with125I-T3. These properties of125I-Triac may be useful in efforts to demonstrate RT in tissues that have low RT levels and/or when serum contamination is present.