The isolation, cloning, and expression of a cDNA insert complementary to mRNA encoding human 3β- hydroxysteroid dehydrogenase/δ5→4isomerase is reported. The insert contains an open reading frame encoding a protein of 372 amino acids, the initial 29 amino acids corresponding to the Nterminal sequence identified from the purified human placental microsomal enzyme. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to human placental microsomal 3β-hydroxysteroid dehydrogenase/ Aδ5→4isomerase, as detected by immunoblot analysis, and catalyzed the conversion of 17α-hydroxypregnenolone to 17α-hydroxyprogesterone, pregnenolone to progesterone, and dehydroepiandro3- terone to androstenedione. Transfected COS cell homogenates, supplemented with NAD⁺, very efficiently oxidized 5α-androstan- 3β,17β-diol to 5α-dihydrotestosterone and, upon addition of NADH, reduced 5α-dihydrotestosterone to 5α-androstan- 3β,17β-diol. Thus, the dehydrogenation/isomerization steps of steroid biosynthesis can be catalyzed by a single polypeptide chain, which can metabolize all of the major physiological substrates. (Endocrinology126: 2493–2498, 1990)