1alpha,25-dihydroxycholecalciferol [1alpha,25(OH)2D3], the active form of cholecalciferol, is a negative growth regulator of breast cancer cells. CYP27B1 is a cytochrome P450-containing hydroxylase expressed in kidney and other tissues that generates 1alpha,25(OH)2D3 from an inactive vitamin D precursor 25-hydroxycholecalciferol [25(OH)D3]. In these studies, we tested the hypothesis that mammary cells express CYP27B1 and locally produce 1alpha,25(OH)2D3, which acts in an autocrine manner to regulate cell turnover. Using Western blot and quantitative real-time PCR, CYP27B1 mRNA and protein were detected in immortalized, nontumorigenic human mammary epithelial cell (HMEC) cultures. Furthermore, HMEC cultures were dose dependently growth inhibited by physiological concentrations of 25(OH)D3, suggesting that CYP27B1 converts this precursor cholecalciferol metabolite to 1alpha,25(OH)2D3, the ligand for the vitamin D receptor (VDR). In support of this suggestion, both 1alpha,25(OH)2D3 and 25(OH)D3 transactivated VDR in HMEC cultures, as measured by induction of a vitamin D responsive reporter gene and upregulation of CYP24, an endogenous VDR target gene. No induction of CYP24 by 25(OH)D3 was observed in mammary cells derived from CYP27B1 null mice. Similar results were observed in 2 independently derived immortalized HMEC lines as well as in primary cultures derived from human breast epithelium. These are the first studies to demonstrate that nontransformed human mammary cells express CYP27B1, that they are growth inhibited by physiologically relevant concentrations of 25(OH)D3, and that they provide a biological mechanism linking vitamin D status to breast cancer risk.