Eleven -hydroxysteroid dehydrogenase type 1, expressed mainly in the endoplasmic reticulum of adipocytes and hepatocytes, plays an important role in the prereceptorial activation of glucocorticoids. In liver endoplasmic reticulum-derived microsomal vesicles, NADPH supply to the enzyme is guaranteed by a tight functional connection with hexose-6-phosphate dehydrogenase and the glucose-6-phosphate transporter, G6PT. In adipose tissue, the proteins and their activities supporting the action of 11-hydroxysteroid dehydrogenase type 1 have not been explored, yet. Here we report the occurrence of the hexose-6-phosphate dehydrogenase in rat epididymal fat, as detected at the level of mRNA, protein, and activity. In the isolated microsomes, the activity was evident only upon the permeabilization of the membrane, because of the poor permeability to the co-factor NADP⁺, which is consistent with the intralumenal compartmentation of both the enzyme and a pool of pyridine nucleotides. In fat cells, the access of the substrate, glucose-6-phosphate to the intralumenal hexose-6-phosphate dehydrogenase appeared to be mediated by the liver-type G6PT. In fact, the G6PT expression was revealed at the level of mRNA and protein. Accordingly, the transport of glucose-6-phosphate was demonstrated in microsomal vesicles, and it was inhibited by S3483, a prototypic inhibitor of G6PT. Furthermore, isolated adipocytes produced cortisol upon addition of cortisone, and the production was markedly inhibited by S3483. The results show that adipocytes are equipped with a functional G6PT - hexose-6-phosphate dehydrogenase - 11-hydroxysteroid dehydrogenase type 1 system, and indicate that all the three components are potential pharmacological targets for modulating local glucocorticoid activation.