In this study we demonstrate the presence of specific, high-affinity receptors for 1,25-dihydroxyvitamin D3 in malignant melanoma. Receptors are present both in cultured melanoma cells and in melanoma tumor tissue produced by inoculation of cells into athymic rats. The receptor sediments at 3.25 on sucrose density gradients, possesses a preferential affinity for 1,25-(OH)2D3 and has an apparent Kd of 0.18 nM by Scatchard analysis. We also demonstrate that human melanoma cells are responsive to 1,25-(OH)2D3 in vitro. Inclusion of 1,25-(OH)2D3 in the culture medium produced a marked increase in cell doubling time. This inhibitory effect of the hormone on melanoma cell proliferation was dose-related and represents the first demonstration of a 1,25-(OH)2D3 mediated action on tumor cells.