Acetoacetate decarboxylase is a valuable tool for clinical analysis of ketone bodies in human plasma. After screening many microorganisms, we found Bacillus polymyxa A-57 to be a new enzyme source with a good yield of acetoacetate decarboxylase. After purifying the intracellular enzyme by three-stage column chromatography, we identified it as a single protein by SDS-polyacrylamide gel electrophoresis. The enzyme had a molecular weight of approximately 280, 000 and consisted of 10 (±2) identical subunits. It had an optimum pH of 5.9 and was stable up to about 60°C for 30min. The apparent Km and Vmax values for lithium acetoacetate were 0.94mM and 296μmol/min/mg, respectively. In addition, the decarboxylase activity was found in the broth. After purification, we found that it was due to an active peptide which we named A-57-9, which we identified as the antibiotic polymyxin M1. However, the Vmax value (0.25μmol/min/mg) of the peptide was very much lower and the Km value (400 mM) was higher than those of real acetoacetate decarboxylase.