The genomic locus containing the potential repressor gene mdcY (inactivated by a putative IS3 element) and the mdcLMACDEGBH genes from Acinetobacter calcoaceticus was cloned and sequenced. In order to evaluate the biochemical function of the protein components, the genes were expressed independently and their activities predicted by database analysis. The mdcA gene product, the alpha subunit, was found to be malonate/acetyl-CoA transferase and the mdcD gene product, the beta subunit, was found to be malonyl-CoA decarboxylase. The mdcE gene product, the gamma subunit, may play a role in subunit interaction to form a stable complex or as a codecarboxylase. The mdcC gene product, the delta subunit, was an acyl-carrier protein, which has a unique CoA-like prosthetic group. Various combinations of malonate decarboxylase subunits allowed us to estimate their contribution to malonyl-CoA decarboxylase activity. The prosthetic group was identified as carboxymethylated 2'-(5"-phosphoribosyl)-3'-dephospho-CoA by mass spectrometry. The mdcH gene product was determined to have malonyl-CoA/dephospho-CoA acyltransferase activity. Using database analysis mdcLM, mdcG, mdcB and mdcI were estimated to be the genes for a malonate transporter, a holo-acyl carrier synthase, protein for the formation of precursor of the prosthetic group and a regulatory protein, respectively. From the data shown above we propose a metabolic pathway for malonate in A. calcoaceticus.