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Regulation of the metabolism of [³H]25-hydroxyvitamin D₃ ([³H]25-(OH)D₃) in vitro to material with the characteristics of [³H]24,25-dihydroxyvitamin D₃ ([³H]24,25-(OH)₂D₃) has been studied in the human promyelocytic cell line HL60. Synthesis of 24,25-(OH)₂D₃ was induced in a dose-dependent manner in cells pretreated with 0·1–100 nM 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) for 4 days. This treatment also inhibited cell proliferation and stimulated differentiation to a macrophage phenotype that was characterized by staining for non-specific esterase (NSE) activity. The ability to synthesize [³H]24,25-(OH)₂D₃ from [³H]25-(OH)D₃ and the expression of NSE activity both responded to changes in concentration of 1,25-(OH)₂D₃ in the culture medium in a parallel manner. Synthesis of [³H]24,25-(OH)₂D₃ was linear when the incubation time was between 1 and 8 h and the cell number between 1 and 12x10⁶ cells/incubation. The optimum substrate concentration for its synthesis was 125 nM, giving an apparent Michaelis constant of 360 nM. The identity of the [³H]24,25-(OH)₂D₃ synthesized by these cells was confirmed by co-chromatography with authentic 24,25-(OH)₂D₃ on normal-phase and reverse-phase high-performance liquid chromatography systems and by its reaction to sodium-m-periodate. Cells that had been exposed to 100 nM 1,25-(OH)₂D₃ for 4 days synthesized 2·17±0·07 (S.E.M.) pmol 24,25-(OH)₂D₃/10⁶ cells per h. This synthesis was inhibited in a dose-dependent manner over a concentration range of 0·01–1 µM by the drug ketoconazole, an antimycotic imidazole which is a known inhibitor of certain cytochrome P-450 enzyme systems, suggesting that the HL60 25-(OH)D₃-24-hydroxylase is also a P-450-dependent enzyme system.