The type 2 isoform of human 17β-hydroxysteroid dehydrogenase (17βHSD2) efficiently catalyzes the oxidative metabolism of androgens and estrogens, and it is expressed in a large series of human peripheral tissues. To obtain a better understanding of the regulation of local steroid biosynthesis and metabolism in human tissues, we have established a dual steroidogenic activity of the 17βHSD2 enzyme after transfection of human 17βHSD2-transfected human embryonic kidney (293) cells. After transient transfection, the metabolism of testosterone, pregnenolone, and dehydroepiandrosterone (DHEA) in intact transfected 293 cells was evaluated by TLC-based radiometric assays. 17βHSD2-transfected cells converted 91% of testosterone (1 μmol/L) into androstenedione in a 2-h incubation period. In addition, pregnenolone (1 μmol/L) was converted to progesterone (18.5%), whereas DHEA (1 μmol/L) was metabolized to androstenedione (8.3% conversion) in a 15-h incubation period. The kinetics of the 3β-hydroxysteroid dehydrogenase (3βHSD) and 17βHSD2 activities using cell homogenate protein of stably transfected 293 cells indicated that the catalytic efficiency (apparent catalytic efficiency = maximum velocity/K(m)) of this 3βHSD activity is approximately 2000-fold (pregnenolone as substrate) or 3000-fold (DHEA as substrate) weaker than that of 17βHSD2 activity. It is noteworthy, however, that the apparent catalytic efficiency of the HSD3B2 gene product is only approximately 50-fold higher than that of the 3βHSD aspect of the 17βHSD2 gene product. Pregnenolone or DHEA effectively inhibited 17βHSD2 activity in a noncompetitive fashion. Furthermore, the potent 5α-reductase/3βHSD inhibitor, 17β-N,N-diethylcarbamoyl-4-methyl-4-aza-5α-androstane-3-one, inhibited neither 3βHSD nor 17βHSD2 activities. We conclude that human 17βHSD2 enzyme exhibits 3βHSD activity. Notwithstanding that this 3βHSD activity is reduced compared to 17βHSD oxidative activity, it may account for at least some of the reports of 3βHSD activity found in human peripheral tissues that express notable amounts of the 17βHSD2 isozyme as well as in individuals with severe classic 3βHSD deficiency.