Malonate kinase is a novel enzyme that catalyzed the formation of malonylphosphate directly from malonate and ATP. The enzyme was induced in malonate-grown Acinetobacter caloaceticus and activated by sulfate ion and by Triton X-100. The enzyme was purified by a combination of ammonium sulfate precipitation, Affi-gel blue, polybuffer exchanger 94, ω-aminohexyl agarose, and hydroxyapatite chromatography. The enzyme was composed of two proteins, protein A and protein B, separated on polybuffer exchanger 94. The protein A was 63,000 dalton monomer and the protein B was 96,000 dalton dimer (2×48,000). This enzyme showed a high substrate specificity on malonate and ATP, and required Mg^(2+) or Mn^(2+). Optimal pH was 7.2.