The FRTL-5 cell line is widely used as a model for normal thyroid follicular cells. These cells have retained their ability to alter cAMP production, cell proliferation, iodine uptake, and thyroglobulin synthesis in response to thyrotropin. We have previously shown that calcitriol attenuated both basal and TSH stimulated cAMP production dose-dependently in FRTL-5 cells. Cytosol fractions (105000 g, 60 min, 4°C) prepared from FRTL-5 cell homogenates possessed calcitriol-binding components with a sedimentation coefficient of approximately 3.7 S in high salt (0.3 mol/l KCl) sucrose gradients (5–20%). At 4°C, specific binding increased rapidly during the first 4 h and reached a plateau after 8 h. The specific binding (18 h, 4°C) was maximal at a [3H]calcitriol concentration of approximately 0.5 nmol/l. Scatchard analysis of the binding data indicated one single class of high affinity binding sites with Kd = 105±2 pmol/l and Bmax=38.5±4.7 pmol/g cytosol protein (mean ± sd, N=6). In conclusion, our results suggest that the FRTL-5 cells possess functional receptors for calcitriol with the same physicochemical properties as the receptors found in normal rat tissues.