A cytochrome P-450 that catalyzes the 24-hydroxylation of 25-hydroxyvitamin D3 (P-450cc24: P-450cholecalciferol24) was purified to electrophoretic homogeneity from the kidney mitochondria of female rats treated with vitamin D3 (Ohyama, Y., Hayashi, S., and Okuda, K. (1989) FEBS Lett. 255, 405-408). The molecular weight was 53,000, and its absorption spectrum showed peaks characteristic of cytochrome P-450. The turnover number was 22 min-1 and the specific content was 2.8 nmol/mg protein. The N-terminal amino acid sequence, Arg-Ala-Pro-Lys-Glu-Val-Pro-Leu-, is different from the N-terminal sequence of any other cytochrome P-450s so far reported. Upon reconstitution with the electron-transferring system of the adrenal mitochondria, the enzyme showed a high activity in hydroxylating 25-hydroxyvitamin D3 as well as 1 alpha,25-dihydroxyvitamin D3 at position 24. However, the purified enzyme hydroxylated neither vitamin D3 nor 1 alpha-hydroxyvitamin D3. The enzyme was also inactive toward xenobiotics. The enzyme hydroxylated 25-hydroxyvitamin D3 at position 24 but not at 1 alpha, indicating that the enzyme is distinct from that catalyzing 1 alpha-hydroxylation. The reaction followed Michaelis-Menten kinetics, and the Km value for 25-hydroxyvitamin D3 was 2.8 microM. Both vitamin D3 and 1 alpha-hydroxyvitamin D3 inhibited the 24-hydroxylation of 25-hydroxyvitamin D3 in a competitive, concentration-dependent manner. 25-Hydroxyvitamin D3 24-hydroxylase activity was significantly inhibited by 7,8-benzoflavone, ketoconazole, and CO, whereas it was only slightly inhibited by aminoglutethimide, metyrapone, and SKF-525A. Mouse antibodies raised against the cytochrome P-450 inhibited the reaction about 70% and reacted with the P-450cc24 in immunoblotting but did not react with other kinds of cytochrome P-450 in rat liver microsomes and mitochondria.