Saturable, high-affinity binding sites for 3,5,3'-triiodo-L-thyronine (T3) were identified in isolated nuclei and solubilized chromatin extracts of mammary glands, spontaneous mammary tumors, and liver from C3H/HeN mice. Receptor concentration in whole mammary gland nuclei (254 fmol/mg DNA) was only about one-half that of mouse liver nuclei (536 fmol/mg DNA), but in molecular weight (55,000) and in their affinity for various thyroid hormone analogues, the binding was essentially identical. Saturation analysis of T3 binding in a series of individual spontaneous mammary tumors and pooled lactating mammary glands indicated that the concentrations of T3-binding sites of the mammary gland are conserved in the transition to neoplasms and are somewhat increased in the largest tumors. Thyroxine binding was identical in capacity to T3 binding in mammary gland nuclei and nuclear extract but showed a higher binding capacity than did T3 in the largest tumors. High-performance molecular exclusion chromatography did show a difference between mammary gland and liver in the distribution of competible [125I]T3 binding between two macromolecular forms; the excluded peak (Mr greater than 450,000) comprised 56% of the T3 binding in the liver but only 9% in the mammary gland with the included peak (Mr 55,000) contributing the balance of binding in each case. Spontaneous mammary tumor resembled the mammary gland in the macromolecular distribution of specific T3 binding (16% excluded). Thymidine uptake showed only a modest decrease in the larger tumors (greater than 2.0 g), while nuclear histone acetylase activity was significantly decreased in this group. Neither measurement showed a significant correlation with T3 or thyroxine binding capacity.