The complete nucleotide sequence of the Saccharomyces cerevisiae CPT1 gene, a structural gene for the sn-1,2-diacylglycerol cholinephosphotransferase (Hjelmstad, R. H., and Bell, R. M. (1987) J. Biol. Chem. 262, 3909-3917), was determined. The 2,100-nucleotide extent of DNA sequenced contained an open reading frame encoding 407 amino acids interrupted by an intron near its 5'-end. Northern hybridization analysis detected the presence of 1.4- and 1.7-kilobase transcripts corresponding to the CPT1 gene. S1 nuclease mapping experiments indicated that the 1.4-kilobase transcript was initiated 80 nucleotides upstream from the translational start site near a poly(dA-dT) promoter element and established that the predicted intron was removed in vivo. The previously constructed cpt1::LEU2 insertional mutation was shown to involve disruption of the CPT1 open reading frame approximately in the middle; this construct did not support the production of a stable transcript. The CPT1 promoter region contained several elements homologous to the promoter regions of other phospholipid biosynthetic structural genes. A model for the membrane topography of the predicted 46,305-dalton cholinephosphotransferase was constructed on the basis of predictive methods. The presence of seven transmembrane helices and an asymmetric distribution of hydrophilic regions were predicted. Regional protein homologies to the acetylcholine receptor, phosphoglycerate kinase, and several cytidine diphosphate utilizing enzymes suggested a functional asymmetry which precisely correlated with the predicted topological asymmetry.