Syringomycin E is an antifungal cyclic lipodepsinonapeptide produced by Pseudomonas syringae pv. syringae. To understand the mechanism of action of syringomycin E, a novel resistant Saccharomyces cerevisiae strain, BW7, was isolated and characterized. Lipid analyses revealed that BW7 contained only the hydrophobic subspecies of sphingolipids that are normally minor components in wild type strains. This aberrant sphingolipid composition was the result of lack of alpha-hydroxylation of the amide-linked very long chain fatty acids, suggesting a defective sphingolipid alpha-hydroxylase encoded by the FAH1 gene. A yeast strain that lacks the FAH1 gene was resistant to syringomycin E, and failed to complement BW7. These results demonstrate that BW7 carries a mutation in the FAH1 gene, and that the lack of alpha-hydroxylated very long chain fatty acids in yeast sphingolipids confers resistance to syringomycin E.