The isolation, cloning, and expression of a cDNA insert complementary to mRNA encoding human 3 beta-hydroxysteroid dehydrogenase/delta 5----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 N-terminal 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 3 beta-hydroxysteroid dehydrogenase/delta 5----4isomerase, as detected by immunoblot analysis, and catalyzed the conversion of 17 alpha-hydroxypregnenolone to 17 alpha-hydroxyprogesterone, pregnenolone to progesterone, and dehydroepiandrosterone to androstenedione. Transfected COS cell homogenates, supplemented with NAD+, very efficiently oxidized 5 alpha-androstan-3 beta,17 beta-diol to 5 alpha-dihydrotestosterone and, upon addition of NADH, reduced 5 alpha-dihydrotestosterone to 5 alpha-androstan-3 beta,17 beta-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.