Intracellular concentrations of the glucocorticoids cortisol and corticosterone are modulated by the enzymes 11ß-hydroxysteroid dehydrogenase 1 and 2 (11ßHSD1 and 11ßHSD2). 11ßHSD1 is an NADPH-dependent microsomal reductase that converts the inactive glucocorticoids cortisone and 11-dehydrocorticosterone to their active forms, cortisol and corticosterone. Hexose-6-phosphate dehydrogenase (H6PDH) is an enzyme that generates NADPH from NADP⁺ within the endoplasmic reticulum. In the absence of NADPH or H6PDH to regenerate NADPH, 11ßHSD1 acts as a dehydrogenase and inactivates glucocorticoids, as does 11ßHSD2. A monoclonal antibody against H6PDH was produced to study the possibility that 11ß -HSD1 in the absence of H6PDH may be responsible for hydroxysteroid dehydrogenase activity in tissues that do not express significant amounts of 11ßHSD2. H6PDH and 11ßHSD1 expression was surveyed in a variety of rat tissues by real time RT-PCR, western blot analysis and immunohistochemistry. H6PDH was found in a wide variety of tissues with the greatest concentrations in the liver, kidney and leydig cells. While the brain as a whole did not express significant amounts of H6PDH, some neurons were clearly immunoreactive by immunohistochemistry. H6PDH was amply expressed in most tissues examined in which 11ßHSD1 was also expressed, with the notable exception of the renal interstitial cells, where dehydrogenase activity by 11ßHSD1 probably moderates activation of the glucocorticoid receptor, as rat renal interstitial cells do not have significant amounts of mineralocorticoid receptors. This antibody against the H6PDH should prove useful for further studies of enzyme activity requiring NADPH generation within the endoplasmic reticulum.