A bacterial strain of Acinetobacter sp., which was capable of enzymatic production of pyruvate from lactate, was cultured in a 5-l reactor with a basal salt medium. After 14 h of fed-batch fermentation, 9.56 g l(-1) cell concentration in the broth was obtained with 20 g l(-1) (178 mM) sodium lactate and 4 g l(-1) NH(4)Cl in the medium; and the biotransformation ability was 2.51 units ml(-1). The cells were harvested from one reactor and then used for pyruvate production from lactate in the same reactor. l-lactate at a concentration about 527 mM was almost stoichiometrically converted to pyruvate in 28 h. After a total 42 h of cell culture and biotransformation, the transformative yield was about 0.72 g g(-1) pyruvate from lactate and the rate of pyruvate production was calculated as 1.33 g l(-1) h(-1) during the process. The results suggested this simple enzymatic production of pyruvate from lactate should be a promising process and may bring a yield higher than that by microbial fermentation. By this process, the recovery of pyruvate from such a simple reaction liquid is relatively easy and inexpensive to perform.