The complete nucleotide sequence of the Saccharomyces cerevisiae EPT1 gene, a structural gene encoding an sn-1,2-diacylglycerol ethanolamine- and cholinephosphotransferase (Hjelmstad, R. H., and Bell, R. M. (1988) J. Biol. Chem. 263, 19748-19757), was determined. The 2123-nucleotide extent of DNA sequenced contained an open reading frame encoding 391 amino acids interrupted by an intron near its 5' end. Northern hybridization analysis detected a single 1.4-kilobase transcript. The inferred 44,525-dalton EPT1 gene product exhibited 54% amino acid sequence homology to the cholinephosphotransferase product of the yeast CPT1 gene. Predictive structural analysis of the EPT1 gene product revealed close structural similarity to the CPT1 gene product with respect to membrane topography, features of secondary structure, and transmembrane asymmetry. Regional protein homologies were identified between the EPT1 gene product and several enzymes as well as the nicotinic acetylcholine receptor. Comparative analysis of this set of protein homologies and the related set of protein homologies to the CPT1 gene product permitted identification of a presumptive active site region which contains highly conserved and divergent subregions and a common mononucleotide binding site.