AbstractAcetobacterpasteurianuswhich is widely used for commercial brewing of vinegar can grow well with lactate as an energy source. In the utilization processes of lactate in A. pasteurianus, it is first converted to pyruvate, and then converted to final product; acetate via acetaldehyde. In the other pathway, pyruvate, formed from lactate is metabolized through the tricarboxylic acid (TCA) cycle. The enzymes catalyzing reaction from pyruvate to acetaldehyde had been identified, however, the enzyme responsible for the oxidation of lactate to pyruvate in A. pasteurianus have not been identified. In this study, we focused on the enzymes involved in the process and aimed at their characterization. This study will contribute to quality improvement of vinegar which provides human health with a good effect. A. pasteurianus assimilated D-lactate as well as L-lactate, indicating that the enzymes catalyzing the oxidation of D- and L-lactate to pyruvate could be produced in the cell. The presence of the enzyme which catalyzes the oxidation of lactate to pyruvate in A. pasteurianus was confirmed by enzymatic assays using dichloropenol-indophenol as redox dye, phenazinemethosulfate as electron acceptor, and lactate as substrate.