2,3-butanediol dehydrogenase (BDH) catalyzes the NAD-dependent redox reaction between acetoin and 2,3-butanediol. There are three types of homologous BDH, each stereospecific for both substrate and product. To establish how these homologous enzymes possess differential stereospecificities, we determined the crystal structure of l-BDH with a bound inhibitor at 2.0 A. Comparison with the inhibitor binding mode of meso-BDH highlights the role of a hydrogen-bond from a conserved Trp residue(192). Site-directed mutagenesis of three active site residues of meso-BDH, including Trp(190), which corresponds to Trp(192) of L-BDH, converted its stereospecificity to that of L-BDH. This result confirms the importance of conserved residues in modifying the stereospecificity of homologous enzymes.