The renal 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 2 catalyzes the NAD(+)-dependent oxidation of the C11-alcohol on cortisol and corticosterone to yield inactive 11-ketosteroids. The lack of purified active enzyme complicates structure-function analyses of 11beta-HSD2. Here, we constructed a 3D-structural model of 11beta-HSD2, based on known 3D-structures of other short-chain dehydrogenases/reductases (SDR), and functionally analyzed 11beta-HSD2 mutants predicted to be involved in cofactor binding. Our 3D-model explains the preference for NAD(+) over NADP(+) by the coulombic repulsion between the adenosine ribose 2'-phosphate on NADP(+) and the carboxylate on Glu(115) and to steric hindrance with the side chain on Glu(115). Indeed, replacement of Glu(115) with serine or threonine, lacking repulsive charge and unfavorable steric interactions, showed only 3-fold preference for NAD(+), compared to 40-fold for wild-type 11beta-HSD2. Mutation of both Asp(91) and Glu(115) to serine raised NADP(+)-dependent activity to that with NAD(+), but caused reduced enzymatic activity. The 3D-model predicted that this is due to a loss of stabilizing interactions of Asp(91) with Cys(90), Glu(115), Asn(117) and Gly(120). Thus, predictions using the 3D-model combined with analysis of mutants allowed the identification of residues critical for NAD(+)-dependent activity of 11beta-HSD2.