The structural gene encoding 25-hydroxyvitamin D3 24-hydroxylase (P-450cc24) was isolated from the rat genomic DNA. It spans approximately 15 kb, is composed of 12 exons, and was demonstrated by Southern blot analysis to be present as a single copy. One major T residue was identified at the cap site, a putative TATA (ATAAATA) box was located at position -30, and a putative CCAAT box was at -58. Four possible vitamin D responsive elements that may be involved in regulation of 24-hydroxylase expression were found in the 5'-flanking region. Alignment with mitochondrial P-450 proteins showed that 7 out of 11 intron insertion sites of P-450cc24 gene occupied positions identical with those in the CYP11 family (P-450scc, P-450(11 beta)). The structure of the gene is discussed in relation to present knowledge about the mechanism of regulation of the 25-hydroxyvitamin D3 24-hydroxylase and calcium homeostasis.