Both cortisol and aldosterone bind to and activate the mineralocorticoid receptor. Cortisol concentrations are generally 100- to 200-fold higher than aldosterone concentrations, yet mineralocorticoids clearly exert effects different from glucocorticoids. One hypothesis is that 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), which converts cortisol to biologically inactive cortisone, protects the mineralocorticoid receptor from cortisol. The circulating concentrations of cortisol in the squirrel monkey are 20- to 50-fold higher than human cortisol concentrations, yet this animal has no evidence of glucocorticoid or mineralocorticoid excess. We used this experiment of nature to test the hypotheses that the known (hepatic) form of 11 beta-HSD protects renal mineralocorticoid receptors from the action of cortisol and that it modulates glucocorticoid concentrations in target tissues. Using a long oligonucleotide based on the rat sequence, we cloned the squirrel monkey 11 beta-HSD complementary DNA and gene. The encoded monkey amino acid sequence is 75% and 91% identical to the corresponding rat and human sequences, respectively. The tissue abundance of the messenger RNA for the monkey enzyme was similar to or less than that seen for the rat and human enzymes. Both the monkey and human 11 beta-HSD complementary DNAs were cloned into an expression vector and used to transfect cultures of Chinese hamster ovary cells. Both vectors were transcribed and translated into equivalent amounts of 11 beta-HSD enzyme. The monkey enzyme was slightly more efficient than the human enzyme in converting [3H]cortisol to cortisone, and estimates of the Michaelis-Menten constant and maximum velocity of both enzymes are similar. These data indicate that the abundance and activity of the hepatic form of 11 beta-HSD are insufficient to inactivate the very high concentrations of cortisol in the squirrel monkey, suggesting that this form of 11 beta-HSD does not defend the mineralocorticoid receptor or protect tissues from high cortisol concentrations. Rather, this enzyme appears to favor conversion of cortisone to cortisol, thus maximizing tissue concentrations of cortisol to overcome glucocorticoid resistance associated with a 50% reduction in glucococorticoid receptors.