A search of the Saccharomyces cerevisiae genome data base for cytochrome b5-like sequences identified a 1.152-kilobase pair open reading frame, located on chromosome XIII at locus YMR272C (FAH1). That gene encodes a putative 384-amino acid protein with an amino-terminal cytochrome b5 domain. The b5 core domain shows a 52% identity and 70% similarity to that of the yeast microsomal cytochrome b5 and a 35% identity and 54% similarity to the b5 core domain of OLE1, the S. cerevisiae Delta-9 fatty acid desaturase. Expression of the S. cerevisiae FAH1 cytochrome b5 domain in Escherichia coli produces a soluble protein that exhibits the typical oxidized versus reduced differential absorbance spectra of cytochrome b5. Sequence analysis of Fah1p reveals other similarities to Ole1p. Both proteins are predicted to have two hydrophobic domains, each capable of spanning the membrane twice, and both have the HX(2-3)(XH)H motifs that are characteristic of membrane-bound fatty acid desaturases. These similarities to Ole1p suggested that Fah1p played a role in the biosynthesis or modification of fatty acids. Disruption of the FAH1 gene in S. cerevisiae did not give any visible phenotype, and there was no observable difference in content or distribution of the most abundant long chain saturated and unsaturated 14-18-carbon fatty acid species. Northern blot analysis, however, showed that this gene is expressed at much lower levels ( approximately 150-fold) than the OLE1 gene, suggesting that it might act on a smaller subset of fatty acids. Analysis of sphingolipid-derived very long chain fatty acids revealed an approximately 40-fold reduction of alpha-HO 26:0 and a complementary increase in 26:0 in the gene-disrupted fah1Delta strain. GAL1 expression of the S. cerevisiae FAH1 genes in the fah1Delta strain restores alpha-HO 26:0 fatty acids to wild type levels. Also identified are a number of homologs to this gene in other species. Expression of an Arabidopsis thaliana FAH1 gene, which does not contain the cytochrome b5 domain, in the fah1Delta strain produced an approximately 25-fold increase in alpha-HO 26:0 and reduced the levels of its 26-carbon precursor, suggesting that it functions in very long chain fatty acid hydroxylation using an alternate electron transfer mechanism.