The antimitotic and prodifferentiating 1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3), synthesized at various extrarenal sites could potentially prevent sporadic tumor development. Physiological regulation of extrarenal Vitamin D hydroxylases following tissue accumulation of 1alpha,25-(OH)2D3 is unknown. We therefore investigated basal and Vitamin D-regulated expression and activity of the synthesizing (CYP27B1) and metabolizing (CYP24A1) hydroxylase in three cell lines derived from the colon, and compared this to cells from the prostate and mammary gland. Our results show that all cells, irrespective of origin and differentiation, express CYP27B1 mRNA, whereas basal CYP24A1 mRNA is highly expressed only in undifferentiated cells. Treatment with 1alpha,25-(OH)2D3 diminishes CYP27B1 and Vitamin D receptor mRNA expression, but elevates CYP24A1 mRNA to equal levels in all cells. As shown by HPLC, CYP27B1 is active only if basal 24-hydroxylation is not maximally functional. In turn, accumulation of 1alpha,25-(OH)2D3 will induce 24-hydroxylation. We conclude that, although extrarenal and renal metabolic pathways for Vitamin D are similar, malignancy of tumor cells determines extent of Vitamin D catabolism.