Cortisol production requires the activity of only 17α -hydroxylase, whereas the formation of sex steroids requires both 17α -hydroxylase and 17,20-lyase activities. Studies in reconstituted enzyme systems have suggested that a single steroid hydroxylase, 17α -hydroxylase cytochrome P-450 (P-45017α), catalyzes both activities. By expression of bovine adrenocortical P-45017α in COS 1 (transformed monkey kidney) cells, which normally contain no detectable P-45017α, it has now been established in situ that a single polypeptide chain does catalyze both the 17α -hydroxylase and the 17,20-lyase reactions. This heterologous system supports 17α -hydroxylation of pregnenolone and progesterone with equal efficiency, but catalyzes about five times as much 17,20-lyase activity when 17α -hydroxypregnenolone is the substrate than when 17α -hydroxyprogesterone is the substrate. For these activities to be observed in COS 1 cells, newly synthesized apocytochrome P-45017α must bind heme and insert into the endoplasmic reticulum such that endogenous cytochrome P-450 reductase can support hydroxylation. Thus, COS 1 cells are a useful system for expression and study of various forms of cytochrome P-450.