Four unlinked fatty acid activation (FAA) genes encoding acyl-CoA synthetases have been identified in Saccharomyces cerevisiae and characterized by noting the phenotypes of isogonic strains containing all possible combinations of faa null alleles. None of these genes is required for vegetative growth when acyl-CoA production by the fatty acid synthetase (Fas) complex is active. When Fas is inhibited by cerulenin, exponentially growing cells are not viable on media containing a fermentable carbon source unless supplemented with fatty acids such as myristate, palmitate, or oleate. The functionally interchangeable FAA1 and FAA4 genes are responsible for activation of these imported fatty acids. Analysis of lysates prepared from isogonic FAA1FAA4 and faa1Δfaa4Δ strains indicated that Faa1p and Faa4p together account for 99% of total cellular myristoyl-CoA and palmitoyl-CoA synthetase activities. Genetic complementation studies revealed that rat liver acyl-CoA synthetase (RLACS) rescues the viability of faa1Δfaa4Δ cells in media containing a fermentable carbon source, myristate or palmitate, plus cerulenin. Rescue is greater at 37 °C compared with 24 °C, paralleling the temperature-dependent changes in RLACS activity in vitro as well as the enzyme's ability to direct incorporation of tritiated myristate and palmitate into cellular phospholipids in vivo. Complementation by RLACS is blocked by treatment of cells with triacsin C (1-hydroxy-3-(E,E,E,2',4',7'- undecatrienylidine)triazene). Even though Faa1p, Faa4p, and RLACS are all able to activate imported myristate and palmitate in S. cerevisiae, the sensitivity of Faa4p and RLACS, but not Faa1p, to inhibition by triacsin C suggests that the rat liver enzyme is functionally more analogous to Faa4p than to Faa1p. Finally, an assessment of myristate and palmitate import into FAA1FAA4 and faa1Δfaa4Δ strains, with or without episomes that direct overexpression of Faa1p, Faa4p or RLACS, indicated that fatty acid uptake is not coupled to activation in S. cerevisiae.