The Saccharomyces cerevisiae CPT1 and EPT1 genes are structural genes encoding distinct sn-1,2-diacylglycerol choline- and ethanolaminephosphotransferases. A haploid cpt1 ept1 double null mutant lacked detectable choline- and ethanolaminephosphotransferase activity but was viable for growth, establishing that these enzymes are nonessential. The activities of the CPT1 and EPT1 gene products were independently studied in membranes prepared from strains mutant in the cognate locus using mixed micellar assays. Both enzymes absolutely required phospholipid cofactors; half-maximal activation was observed at low mole fractions, suggesting that a small number of phospholipid molecules are required. The activities of the CPT1 and EPT1 gene products were compared with respect to dioleoylglycerol dependence, CDP-aminoalcohol specificity, phospholipid activation, and inhibition by CMP. The EPT1 gene product utilized CDP-ethanolamine, -monomethylethanolamine, -dimethylethanolamine, and -choline to significant extents, while the CPT1 gene product manifested relative specificity for CDP-choline and -dimethylethanolamine. The CPT1 and EPT1 gene products exhibited differing properties with respect to phospholipid activation, but this difference was dependent on the CDP-aminoalcohol substrate. In contrast, the two enzymes could be distinguished on the basis of their dioleoylglycerol dependencies, activation by Mg2+, and CMP inhibition profiles regardless of the CDP-aminoalcohol substrate employed. These studies provide the first definitive kinetic properties of individual choline- and ethanolaminephosphotransferases.