The isolation of malonyl-coenzyme A synthetase from Pseudomonas fluorescens grown on malonate has been reported recently (Kim, Y.S., and Bang, S.K. (1985) J. Biol.Chem. 260, 5098-5104). This enzyme is phosphorylated in the presence of ATP and Mg2+. The phosphoryl group appears on one subunit of the enzyme composed of two different subunits, and the phosphoryl enzyme is acid labile and base stable. The phosphoryl group on the enzyme is released by the incubation of the phosphoryl enzyme with malonate and malonyl enzyme is formed. The malonyl enzyme is acid labile and also relatively unstable under basic conditions. The malonyl group is found on the subunit of the enzyme which is phosphorylated. Malonyl-CoA is formed when malonyl enzyme reacts with coenzyme A. These results suggest that two convalent intermediates, phosphoryl and malonyl enzyme, are sequentially formed in the synthesis of malonyl-coenzyme A by malonyl-coenzyme A synthetase catalysis.