Rat liver 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD or AKR1C9), a member of the aldo-keto reductase (AKR) superfamily, plays a pivotal role in the inactivation of circulating steroid hormones. It is the most thoroughly characterized HSD of the AKR superfamily and can be used as a template for structure-function studies in other AKR members such as rodent and human 3alpha-, 17beta- and 20alpha-HSDs. Based on the crystal structure of the E.NADP(+) testosterone ternary complex, there are ten residues that line the testosterone binding cavity: T24, L54, Y55, H117, F118, F129, T226, W227, N306 and Y310. Each residue in the cavity, except for the catalytic residues Y55 and H117, was systematically mutated to alanine to determine the role of the individual residues in steroid recognition. Binding data and kinetic parameters (K(d), k(cat), K(m) and k(cat)/K(m)) of the homogeneous mutants were compared with that of the wild type (WT) enzyme. Titration of the intrinsic tryptophan fluorescence with NADPH demonstrated that cofactor binding was unaltered. However, binding of the steroid hormones testosterone and progesterone to the E.NADPH binary complex was affected to varying degrees. The largest effects on K(d) were an 8-fold decrease in affinity for testosterone and a 50-fold decrease in affinity for progesterone. The mutants bound both hormones in the same rank-order except for W227A, where the binding of progesterone was more adversely affected. A series of 3alpha-hydroxysteroid substrates (A/B trans- and cis-ring fused C(19) and C(21) steroids) were used to determine the ability of each mutant to catalyze steroid turnover. The alanine mutants that retained k(cat)/K(m) values similar to WT were those in which alanine substituted short polar residues such as T24A and T226A. The mutants with the lowest catalytic efficiencies were those in which alanine substituted aromatic residues such as W227A and F129A. The loss in catalytic efficiency was due to large changes in k(cat) (up to 1000-fold), but not K(m). Molecular modeling of the alanine mutants showed that changes in the reaction trajectory defined by the angles and distances by groups that participate in catalysis correlate with changes in k(cat). These results highlight the importance of steroid binding site residues in dictating the proper orientation of substrates to achieve high catalytic turnover while having minimal effects on hormone affinity.