Long chain fatty acids are converted to acyl-CoAs by acyl-CoA synthetase (fatty acid CoA ligase: AMP forming, E.C. 6.2.1.3; ACS). Escherichia coli has a single ACS, FadD, that is essential for growth when fatty acids are the sole carbon and energy source. Rodents have five ACS isoforms that differ in substrate specificity, tissue expression, and subcellular localization and are believed to channel fatty acids toward distinct metabolic pathways. We expressed rat ACS isoforms 1-5 in an E. coli strain that lacked FadD. All rat ACS isoforms were expressed in E. coli fadD or fadDfadR and had ACS specific activities that were 1.6-20-fold higher than the wild type control strain expressing FadD. In the fadD background, the rat ACS isoforms 1, 2, 3, 4 and 5 oxidized [(14)C]oleate at 5 to 25% of the wild type levels, but only ACS5 restored growth on oleate as the sole carbon source. To ensure that enzymes of beta-oxidation were not limiting, assays of ACS activity, beta-oxidation, fatty acid transport, and phospholipid synthesis were also examined in a fadD fadR strain, thereby eliminating FadR repression of the transporter FadL and the enzymes of beta-oxidation. In this strain, fatty acid transport levels were low but detectable for ACS1, 2, 3, and 4 and were nearly 50% of wild type levels for ACS5. Despite increases in beta-oxidation, only ACS5 transformants were able to grow on oleate. These studies show that although ACS isoforms 1-4 variably supported moderate transport activity, beta-oxidation, and phospholipid synthesis and although their in vitro specific activities were greater than that of chromosomally encoded FadD, they were unable to substitute functionally for FadD regarding growth. Thus, membrane composition and protein-protein interactions may be critical in reconstituting bacterial ACS function.