Biochemical and genetic evidence is presented to demonstrate that the prpE gene of Salmonella typhimurium encodes propionyl-CoA synthetase, an enzyme required for the catabolism of propionate in this bacterium. While prpE mutants used propionate as carbon and energy source, prpE mutants that lacked acetyl-CoA synthetase (encoded by acs) did not, indicating that Acs can compensate for the lack of PrpE in prpE mutants. Cell-free extracts enriched for PrpE catalysed the formation of propionyl-CoA in a propionate-, ATP-, Mg2+- and HS-CoA dependent manner. Acetate substituted for propionate in the reaction at 48% the rate of propionate; butyrate was not a substrate for PrpE. The propionyl-CoA synthetase activity of PrpE was specific for ATP. GTP, ITP, CTP and TTP were not used as substrates by the enzyme. UV-visible spectrophotometry, HPLC and MS data demonstrated that propionyl-CoA was the product of the reaction catalysed by PrpE.