In Saccharomyces cerevisiae Fat1p and fatty acyl-CoA synthetase (FACS) are hypothesized to couple import and activation of exogenous fatty acids by a process called vectorial acylation. Molecular genetic and biochemical studies were used to define further the functional and physical interactions between these proteins. Multicopy extragenic suppressors were selected in strains carrying deletions in FAA1 and FAA4 or FAA1 and FAT1. Each strain is unable to grow under synthetic lethal conditions when exogenous long-chain fatty acids are required, and neither strain accumulates the fluorescent long-chain fatty acid C(1)-BODIPY-C(12) indicating a fatty acid transport defect. By using these phenotypes as selective screens, plasmids were identified encoding FAA1, FAT1, and FAA4 in the faa1Delta faa4Delta strain and encoding FAA1 and FAT1 in the faa1Delta fat1Delta strain. Multicopy FAA4 could not suppress the growth defect in the faa1Delta fat1Delta strain indicating some essential functions of Fat1p cannot be performed by Faa4p. Chromosomally encoded FAA1 and FAT1 are not able to suppress the growth deficiencies of the fat1Delta faa1Delta and faa1Delta faa4Delta strains, respectively, indicating Faa1p and Fat1p play distinct roles in the fatty acid import process. When expressed from a 2-mu plasmid, Fat1p contributes significant oleoyl-CoA synthetase activity, which indicates vectorial esterification and metabolic trapping are the driving forces behind import. Evidence of a physical interaction between Fat1p and FACS was provided using three independent biochemical approaches. First, a C-terminal peptide of Fat1p deficient in fatty acid transport exerted a dominant negative effect against long-chain acyl-CoA synthetase activity. Second, protein fusions employing Faa1p as bait and portions of Fat1p as trap were active when tested using the yeast two-hybrid system. Third, co-expressed, differentially tagged Fat1p and Faa1p or Faa4p were co-immunoprecipitated. Collectively, these data support the hypothesis that fatty acid import by vectorial acylation in yeast requires a multiprotein complex, which consists of Fat1p and Faa1p or Faa4p.