Intracellular glucocorticoid reactivation is catalyzed by 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1), which functions predominantly as a reductase in cells expressing hexose-6-phosphate dehydrogenase (H6PDH). We recently showed that the ratios of cortisone to cortisol and 7-keto- to 7-hydroxy-neurosteroids are regulated by 11beta-HSD1 and very much depend on coexpression with H6PDH, providing cosubstrate NADPH. Here, we investigated the impact of H6PDH on the modulation of 11beta-HSD1-dependent interconversion of cortisone and cortisol by inhibitors and alternative substrates. Using HEK-293 cells expressing 11beta-HSD1 or coexpressing 11beta-HSD1 and H6PDH, we observed significant differences of 11beta-HSD1 inhibition by natural and pharmaceutical compounds as well as endogenous hormone metabolites. Furthermore, we show potent and dose-dependent inhibition of 11beta-HSD1 by 7-keto-DHEA in differentiated human THP-1 macrophages and in HEK-293 cells overexpressing 11beta-HSD1 with or without H6PDH. In contrast, 7-ketocholesterol (7-KC) did not inhibit 11beta-HSD1 in HEK-293 cells, even in the presence of H6PDH, but inhibited 11beta-HSD1 reductase activity in differentiated THP-1 macrophages (IC(50) 8.1+/-0.9microM). 7-Keto-DHEA but not 7-KC inhibited 11beta-HSD1 in HEK-293 cell lysates. In conclusion, cellular factors such as H6PDH can significantly modulate the effect of inhibitors and alternative 7-oxygenated substrates on intracellular glucocorticoid availability.