Previous studies have demonstrated that certain 17- and 20-ketosteroids are potent inhibitors of the NADP-linked oxidation of glucose 6-phosphate by mammalian glucose 6-phosphate dehydrogenases. This inhibition is uncompetitive with respect to both NADP+ and glucose 6-phosphate. In order to elucidate the detailed mechanism of this rare type of inhibition, we examined the effects of steroids on human glucose 6-phosphate dehydrogenase catalyzing the reverse reaction, i.e., the reduction of the gluconolactone by NADPH. To circumvent problems associated with the known instability of 6-phospho-delta-gluconolactone, the natural product of glucose 6-phosphate oxidation, the more stable 6-phospho-gamma-gluconolactone was used. Dehydroepiandrosterone, epiandrosterone, 16 alpha-bromoepiandrosterone, and dehydroepiandrosterone sulfate all inhibited the reverse reaction uncompetitively with respect to both NADPH and the gamma-lactone. The Ki values for each of these steroids, determined by varying either coenzyme or substrate in both the forward and the reverse reactions, are very similar. These results demonstrate that steroids inhibit glucose 6-phosphate dehydrogenase by binding to the ternary enzyme-coenzyme-substrate ternary complex(es). This is the first direct demonstration that uncompetitive inhibition of a two-substrate enzyme, by compounds other than its substrates or products, can occur by binding of the inhibitor to a ternary enzyme complex.