Extrarenal 25-hydroxyvitamin D3-1α-hydroxylase is believed to play a major role in the pathogenesis of hypercalcemia associated with various types of granulomatous and lymphoproliferative diseases and certain solid tumors. In this paper, we describe the cloning of the cytochrome P450 component of the extrarenal enzyme from a human nonsmall cell lung carcinoma, SW 900. The cytochrome P450 for the extrarenal 1α-hydroxylase has an amino acid sequence identical to that of the cytochrome P450 component of the CYP1α, the renal form of the enzyme, and appears to be a product of the same gene. CYP1α messenger RNA (mRNA) and 1α-hydroxylase enzyme activity were detected in two (SW 900, SK-Luci-6) of a series of five nonsmall cell lung carcinoma cell lines. All five lung cell lines were cultured with the same medium under the same conditions, but only two of the five expressed 1α-hydroxylase enzyme; two others (WT-E, Calu-1) expressed high levels of the reciprocally regulated enzyme, 25-hydroxyvitamin D3-24-hydroxylase, with its specific cytochrome P450 component, CYP24. Although under basal conditions the lung cell line SW 900 expressed only CYP1α and showed 1α-hydroxylase enzyme activity, when treated with small concentrations of 1α,25-dihydroxyvitamin D3 or high concentrations of 25-hydroxyvitamin D3, it began to express CYP24 and exhibit 24-hydroxylase enzyme activity. Somewhat surprisingly, SW 900 cells still had detectable CYP1α mRNA some 24 h after vitamin D treatment despite the fact that 1α-hydroxylase enzyme activity was unmeasurable. These data are consistent with the emerging hypothesis that vitamin D through its active form does not directly turn off CYP1α mRNA production but, rather, strongly stimulates CYP24, thereby masking CYP1α activity. The factor(s) responsible for the basal expression of CYP1α in SW 900 and SK-Luci-6 is currently unknown.