On an industrial scale, the production of pyruvate at a high concentration from the cheaper lactate substrate is a valuable process. To produce pyruvate from lactate by whole cells, various lactate-utilizing microorganisms were isolated from soil samples. Among them, strain WLIS, identified as Acinetobacter sp., was screened as a pyruvate producer. For the pyruvate preparation from lactate, the preparative conditions were optimized with whole cells of the strain. The cells cultivated in the medium containing 100mM of L-lactate showed the highest biotransformation efficiency from lactate to pyruvate. The optimized dry-cell concentration, pH, and temperature of reaction were 6g/L, pH 7.0-7.5, and 30℃, respectively. The influences of ethylenediaminetetraacetic acid (EDTA) and aeration on a biotransformation reaction were carried out under the test conditions. Under the optimized reaction conditions, L-lactate at concentrations of 200 and 500mM were almost totally stoichiometrically converted into pyruvate in 8 and 12h, respectively. About 60% of 800mM of L-lactate was transformed into pyruvate in 24h. This reduced conversion rate is probably due to the high substrate inhibition in biotransformation.