11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) converts inactive glucocorticoid to their active form (cortisone to cortisol in humans, 11-dehydrocorticosterone (11-DHC) to corticosterone in mice), and is dependent upon the presence of cofactor NADPH generated by the enzyme hexose-6-phosphate (H6PDH) for its activity. The 11β-HSD1/H6PDH system is implicated in the pathogenesis of the metabolic syndrome by generating tissue specific glucocorticoid excess. The use of selective 11β-HSD1 inhibitors therefore offers a novel therapeutic approach. By breeding 11β-HSD1/H6PDH double heterozygous mice on a mixed C57BL/6:129 background we have generated 11β-HSD1−/−/ H6PDH−/− double KO and all conceivable intermediate genotypes (n=5) and assessed their urinary steroid metabolome using GC/MS. This enables us to further understand the role of these enzymes in glucocorticoid metabolism and hypothalamo-pituitaryadrenal axis drive. In H6PDH−/− mice the direction of 11β-HSD1 is reversed and glucocorticoid is inactivated, whereas 11β-HSD1−/− mice only fail to reactivate. Both 11β-HSD1−/− and H6PDH−/− mice have reduced corticosterone metabolites compared to WT animals (% corticosterone metabolites: (WT) 96.1 (11β-HSD1−/−), 67.8 (H6PDH−/−) 2.6), with the levels from H6PDH−/− mice being more exaggerated, consistent with increased inactivation of corticosterone. Double KO mice exhibited similar levels to 11β-HSD1−/− mice, consistent with lack of 11β-HSD1 (% corticosterone metabolites: (11β-HSD1−/−/H6PD−/−) 58.2). Interestingly, urinary metabolites from the intermediate genotypes 11β-HSD1−/+, H6PDH−/+ and 11β-HSD1−/+/ H6PDH−/+ were comparable to those of WT mice (% corticosterone metabolites: (11β-HSD1−/+) 96.8 (H6PDH+/−), 98.8 (11β-HSD1−/+H6PDH−/+) 100.0). Therefore, loss of one allele from either 11β-HSD1 and/or H6PDH does not alter the urinary steroid profile of 11-DHC and corticosterone in mice. Further studies are required to determine the relationship between 11β-HSD1 gene expression and urinary metabolites, and whether this can be applied as a therapeutic tool to assess levels of 11β-HSD1 enzyme activity.