ABSTRACT. Using full length CDNAintroduced into COS-7 cells, two species of P-450 with entirely different physiological functions have been expressed in enzymatically active form. One is P-450d, which is known to reside in the microsomes of rat hepatocytes where it acts as a drug-metabolizing enzyme; the other is P-450(SCC), which catalyzes the conversion of cholesterol to pregnenolone in the rate-limiting reaction of steroidogenesis in mitochondria of adrenal cortex cells. Northern blot and immunoblot analyses revealed that the mRNA and protein of these P-450 species were efficiently produced in COS-7cells. The protein contents amounted to nearly 0.1% of the total cell protein as estimated from immunoblotting and low temperature CO difference spectra. The subcellular localization of the products indicated that they were correctly sorted to the microsomes and mitochondria, respectively. Wehave succeeded in eliciting most of the activity of the expressed microsomal P-450d by reconstruction with NADPH-cytochromeP-450 reductase, while the optimal conditions for the mitochondrial enzyme in the COScells remain to be studied. These results show the applicability of the COS-7expression system to investigations of the functions of members of the P-450 super family whose CDNAhas been newly isolated.Cytochrome P-450 constitutes a super family of hemoproteins which are believed to have evolved from a commonancestor. They catalyze the monooxygenase reaction of xenobiotics, such as drugs, carcinogens and environmental pollutants, as well as endobiotics, such as steroids, fatty acids, and prostaglandins (1, 2). This versatility of the enzyme system is probably due to the large number of P-450 species and their different and sometimes broad substrate specificities. The multiple molecular forms of P-450 have several properties in common,such as size, structure, and immunologicand biochemical characteristics, which makers it difficult to separate one form from another biochemically. Recent application of molecular cloning technology to the field of P-450 has overcome this difficulty and brought about clear-cut identification of many forms of P-450 (2, 3).However, there seem to remain a considerable number of uncharacterized P-450 species, especially the minor forms in higher organisms. Whena CDNA clone for a certain species of P-450 whose function is not knownis isolated by cross-hydridization with the CDNA of a * To whom correspondence and reprint requests should be addressed.known P-450, it is prerequisite for characterization of the newspecies to establish a suitable expression system for the CDNA information. The expression system may also be helpful for investigating the structure-function relationship in P-450 molecules by site-directed mutagenesis and for analyzing causative mutations in defective genes of genetic deseases related to the P-450 superfamily.In the present study we attempted to express two P- molecules expressed in COScells exhibited their correct cellular localizations and functional activities, although th...