A cDNA encoding 25-hydroxyvitamin D3 24-hydroxylase (P450cc24) was isolated from a rat kidney cDNA library using specific antibodies to the enzyme. The isolated cDNA was 3.2 kbp long and contained a 1542-bp open reading frame encoding 514 amino acids. The deduced amino acid sequence contained a presequence typical of mitochondrial enzymes in the N-terminal region. The amino acid sequence shows less than 30% similarity to those of any other cytochrome P450s so far reported and, therefore, P450cc24 constitutes a novel family of P450. COS-7 cells transfected with the cDNA produced a protein that was reactive with the antibodies and catalyzed NADPH-dependent 24-hydroxylation of 25-hydroxyvitamin D3 in the presence of adrenodoxin and NADPH-adrenodoxin reductase. Using the cDNA as a probe we demonstrated that the increase of 24-hydroxylation activity caused by administration of vitamin D3 into rats was accompanied by an increase of the mRNA.