When FRTL-5 cell cytosol was incubated with increasing amounts of [3H]1,25-dihydroxy-vitamin D3 [( 3H]1,25-(OH)2D3), saturation of specific hormone binding occurred. Scatchard analysis of specific binding of [3H]1,25-(OH)2D3 to the macromolecule yielded an apparent Kd value of 0.41 +/- 0.08 X 10(-10) M and a single maximum binding capacity of 42.8 +/- 8.8 fmol/mg protein. Sucrose gradient analysis revealed substantial [3H]1,25-(OH)2D3 association with a macromolecule sedimentating slightly faster than ovalbumin (3.7 S). [3H]1,25-(OH)2D3 was completely displaced by excess 1,25-(OH)2D3. The 1,25-(OH)2D3-receptor complex bound to DNA cellulose columns in low salt buffer, and eluted as a single peak at 0.15-0.20 M KCl. Thus, we have shown for the first time the existence of a functional 1,25-(OH)2D3 receptor in thyroid follicular cells. Furthermore, 1,25-(OH)2D3 inhibited the thyrotropin (TSH)-stimulated iodide uptake in a dose-dependent manner, indicating that 1,25-(OH)2D3 has an effect on the physiological function of rat thyroid follicular cells in culture.