Human 17-hydroxysteroid dehydrogenase types 1 and 2 (17HSD1, 17HSD2) regulate estrogen potency by catalyzing the interconversion of estrone (E1) and estradiol (E2) using nicotinamide adenine dinucleotide (phosphate) cofactors NAD(P)(H). In intact cells, 17HSD1 and 17HSD2 establish pseudo-equilibria favoring E1 reduction or E2 oxidation, respectively. The vulnerability of these equilibrium steroid distributions to mutations and to altered intracellular cofactor abundance and redox state, however, is not known. We demonstrate that the equilibrium E2:E1 ratio achieved by 17HSD1 in intact HEK-293 cell lines is progressively reduced from 94:6 to 10:90 following mutagenesis of R38, which interacts with the 2'-phosphate of NADP(H), and by glucose deprivation, which lowers the NADPH/NADP⁺ ratio. The shift to E2 oxidation parallels changes in apparent K_m values for purified 17HSD1 proteins to favor NAD(H) over NADP(H). In contrast, mutagenesis of E116 (corresponding to R38 in 17HSD1) and changes in intracellular cofactor ratios do not alter the >90:10 E1:E2 ratio catalyzed by 17HSD2, and these mutations lower the apparent K_m of recombinant 17HSD2 for NADP(H) only <3-fold. We conclude that the equilibrium E1:E2 ratio maintained by human 17HSD1 in intact cells is governed by NADPH saturation, which is strongly dependent on both R38 and high intracellular NADPH/NADP⁺ ratios. In contrast, the preference of 17HSD2 for E2 oxidation strongly resists alteration by genetic and metabolic manipulations. These findings suggest that additional structural features, beyond the lack of a specific arginine residue, disfavor NADPH binding thus and support E2 oxidation by 17HSD2 in intact cells.