The Saccharomyces cerevisiae FAT1 gene appears to encode an acyl-CoA synthetase that is involved in the regulation of very long chain (C20-C26) fatty acids. Fat1p, has homology to a rat peroxisomal very long chain fatty acyl-CoA synthetase. Very long chain acyl-CoA synthetase activity is reduced in strains containing a disrupted FAT1 gene and is increased when FAT1 is expressed in insect cells under control of a baculovirus promoter. Fat1p accounts for approximately 90% of the C24-specific acyl-CoA synthetase activity in glucose-grown cells and approximately 66% of the total activity in cells grown under peroxisomal induction conditions. Localization of functional Fat1p:green fluorescent protein gene fusions and subcellular fractionation of C24 acyl-CoA synthetase activities indicate that the majority of Fat1p is located in internal cellular locations. Disruption of the FAT1 gene results in the accumulation of very long chain fatty acids in the sphingolipid and phospholipid fractions. This includes a 10-fold increase in C24 acids and a 6-fold increase in C22 acids. These abnormal accumulations are further increased by perturbation of very long chain fatty acid synthesis. Overexpression of Elo2p, a component of the fatty acid elongation system, in fat1Delta cells causes C20-C26 levels to rise to approximately 20% of the total fatty acids. These data suggest that Fat1p is involved in the maintenance of cellular very long chain fatty acid levels, apparently by facilitating beta-oxidation of excess intermediate length (C20-C24) species. Although fat1Delta cells were reported to grow poorly in oleic acid-supplemented medium when fatty acid synthase activity is inactivated by cerulenin, fatty acid import is not significantly affected in cells containing disrupted alleles of FAT1 and FAS2 (a subunit of fatty acid synthase). These results suggest that the primary cause of the growth-defective phenotype is a failure to metabolize the incorporated fatty acid rather than a defect in fatty acid transport. Certain fatty acyl-CoA synthetase activities, however, do appear to be essential for bulk fatty acid transport in Saccharomyces. Simultaneous disruption of FAA1 and FAA4, which encode long chain (C14-C18) fatty acyl-CoA synthetases, effectively blocks the import of long chain saturated and unsaturated fatty acids.