The role of cholecalciferol, 25(OH) D3, and 1,25(0Hl2 D.3, as modulators of melanocyte function and proliferation has been examined. Topical application of 100 p, g cholecalciferol to the pinnal epidermis of DBA/ZJ mice for 5 or 10 days increased the number of L-dihydroxyphenylalanine-positive (DOPA-positive) melanocytes and had a synergistic effect with a low dose of ultraviolet B light (UVB). Application of 1 pg 1,25(OH):! D3 had a transient effect on epidermal melanocytes. Addition of cholecalciferol to pure cultures of human melanocytes did not alter their tyrosinase activity (therefore, melanin synthesis) or growth rate even after 72 hours of treatment. However, treatment of similar cultures with 1,25(OH)* [I, at a concentration equal to or greater than lo-* M suppressed tyrosinase activity but did not affect proliferation. The effect of 25(OH) D, was similar to, but lower in magnitude than, that of 1 ,25(OHI2 D3. We attempted to demonstrate the presence of specific receptors for 1,25(OH)* D3 in normal human melanocytes using the monoclonal antibody (Mo Ab) 9A7y raised against the receptor for 1,25(OH)2 D3. Melanocytes were exposed to 9A7y and to a secondary biotinylated Ab and analyzed by the fluorescence activated cell sorter (FACS). An increase in the specific fluorescent signal was constantly observed. By using the immunoblotting technique, we observed a major immunoreactive species that migrated in the 53-kD region in normal melanocytes. The size of this major irnmunoreactive species was smaller in melanoma cells than in normal melanocytes. This correlates with the finding that the former cells were unresponsive to cholecalciferol, 25(OH) D3, or 1 ,25(0HI2 D3 treatment. These results predict a direct role for 1,25(OHI2 D3 as an effector of normal melanocyte function.