Two His residues, His-205 and His-296, in Lactobacillus plantarum D-lactate dehydrogenase are highly conserved in the D-isomer-specific 2-hydroxyacid dehydrogenase family, suggesting that they are candidates for the catalytic His in the enzyme. The substitution of His-296 with Tyr by means of site-directed mutagenesis induced a drastic decrease in the reaction rate, while a His-205 to Tyr substitution induced no large change in the catalytic properties. In pyruvate reduction, the Tyr-296 enzyme showed a slightly increased Km below pH 6.0 but no significant pH dependence above pH 6.0, where the wild-type enzyme showed an increased Km value. In D-lactate oxidation, in contrast, the Tyr-296 enzyme showed a greatly increased Km value for D-lactate and strong pH dependence. An additional substitution of His-296 with Gln induced more complete loss of the catalytic activity. In contrast to the Tyr-296 enzyme, the Gln-296 enzyme showed a greatly increased Km value and a strong pH-dependent reaction rate even in pyruvate reduction. Unlike the wild-type or His-205 enzyme, both the Tyr-296 and Gln-296 enzymes showed significant resistance against diethyl pyrocarbonate. These results clearly indicate that His-296 is essential for the catalysis by D-lactate dehydrogenase, as in the case of His-195 in L-lactate dehydrogenase.