The stereochemistry of 2,3-butanediol (2,3-BD) synthesis in microbial fermentations is important for many applications. In this work, we showed that Corynebacterium glutamicum endowed with the Lactococcus lactis genes encoding alpha-acetolactate synthase and decarboxylase activities produced meso-2,3-BD as the major end product, meaning that (R)-acetoin is a substrate for endogenous 2,3-butanediol dehydrogenase (BDH) activity. This is curious in view of the reported absolute stereospecificity of C. glutamicum BDH for (S)-acetoin (Takusagawa et al. Biosc Biotechnol Biochem 65:1876-1878, 2001). To resolve this discrepancy, the enzyme encoded by butA (Cg) was produced in Escherichia coli and purified, and the stereospecific properties of the pure protein were examined. Activity assays monitored online by H-1-NMR using racemic acetoin and an excess of NADH showed an initial, fast production of (2S,3S)-2,3-BD, followed by a slow (similar to 20-fold lower apparent rate) formation of meso-2,3-BD. Kinetic parameters for (S)-acetoin, (R)-acetoin, meso-2,3-BD and (2S,3S)-BD were determined by spectrophotometric assays. V (max) values for (S)-acetoin and (R)-acetoin were 119 +/- 15 and 5.23 +/- 0.06 mu mol min(-1) mg protein(-1), and K (m) values were 0.23 +/- 0.02 and 1.49 +/- 0.07 mM, respectively. We conclude that C. glutamicum BDH is not absolutely specific for (S)-acetoin, though this is the preferred substrate. Importantly, the low activity of BDH with (R)-acetoin was sufficient to support high yields of meso-2,3-BD in the engineered strain C. glutamicum Delta aceE Delta pqo Delta ldhA(pEKEx2-als,aldB,butA (Cg) ). Additionally, we found that the BDH activity was nearly abolished upon inactivation of butA (Cg) (from 0.30 +/- 0.03 to 0.004 +/- 0.001 mu mol min(-1) mg protein(-1)), indicating that C. glutamicum expresses a single BDH under the experimental conditions examined.