We examined the expression and activity of two enzymes from the aldoketoreductase (AKR) family 1C, namely type 5 17beta-hydroxysteroid dehydrogenase (17beta-HSD-5, AKR1C3) and type 3 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD-3, AKR1C2) in female sc and omental adipose tissue and in preadipocyte primary cultures. 17beta-HSD-5 preferentially synthesizes testosterone from the inactive adrenal precursor androstenedione, whereas 3alpha-HSD-3 inactivates dihydrotestosterone. mRNAs of both enzymes were detected in adipose tissue from the omental and sc compartments. Real-time PCR quantification indicated a 3-fold higher 3alpha-HSD-3 expression compared with 17beta-HSD-5, and the expression of both enzymes tended to be higher in the sc vs. the omental depot. Accordingly, dose-response and time-course experiments performed in preadipocyte primary cultures indicated that 3alpha-HSD activity was higher than 17beta-HSD activity (13-fold maximum velocity difference). We measured 3alpha-HSD activity in omental and sc adipose tissue samples of 32 women for whom body composition and body fat distribution were evaluated by dual-energy x-ray absorptiometry and CT, respectively. We found that androgen inactivation in omental adipose tissue through 3alpha-HSD activity was significantly higher in women with elevated vs. low visceral adipose tissue accumulation (1.7-fold difference; P < 0.05). Moreover, omental adipose tissue 3alpha-HSD activity was positively and significantly associated with CT-measured visceral adipose tissue (r = 0.43; P < 0.02) and omental adipocyte diameter (r = 0.42; P < 0.02). These results indicate that local androgen inactivation is a predominant reaction in female abdominal adipose tissue, with the greatest conversion rates observed in the presence of abdominal visceral obesity. Increased androgen inactivation in omental adipose tissue of abdominally obese women may impact locally on the regulation of adipocyte metabolism.