Dehydroepiandrosterone (DHEA) and its sulphate ester DHEAS, have been shown to oppose the effects of glucocorticoids in vivo, thus producing beneficial effects on insulin sensitivity in rodent models of diabetes and obesity as well as in hypoadrenal patients. Glucocorticoids play a key role in regulating fat metabolism and distribution and are reactivated locally by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) oxoreductase activity, which increases with adipocyte differentiation. DHEA has recently been shown to inhibit differentiation and 11β-HSD1 oxoreductase activity in murine 3T3-L1 adipocytes, with the underlying mechanism remaining elusive. Here we have examined the differential effects of DHEA and DHEAS in the human preadipocyte cell line Chub-s7 that stably expresses 11β-HSD1. RT-PCR analysis of Chub-S7 and normal human subcutaneous and omental preadipocytes revealed expression of OATP-C and OATP-D, members of the organic anion transporter peptide family required for the active cross-membrane influx transport of DHEAS but not DHEA. Preadipocyte proliferation as analysed by 3H-thymidine incorporation was significantly inhibited by DHEA (≥10 μM) whilst DHEAS failed to produce an effect. Similarly, preadipocyte differentiation was significantly inhibited by DHEA (≥25 μM), but not by DHEAS, as assessed by cell morphology and mRNA expression of markers of early (LPL) and terminal (G6PDH) differentiation. Furthermore, 11β-HSD1 oxoreductase activity in differentiated adipocytes was significantly inhibited by DHEA (≥10 μM). In addition, DHEA increased insulin-independent glucose uptake in differentiated adipocytes in a dose-dependent fashion. Enzymatic activity assays revealed significant conversion of DHEA to androstenediol in fully differentiated adipocytes but not in preadipocytes, indicating that either of these steroids could be accountable for the observed inhibitory effects upon differentiation. This further highlights the significance of our previous finding that the enzyme responsible for this conversion, 17β-hydroxysteroid dehydrogenase type 5, increases with adipocyte differentiation (JoE 2004, 183:331).