The Saccharomyces cerevisiae CPT1 and EPT1 genes represent structural genes that encode distinct choline- and choline/ethanolaminephosphotransferases, respectively. To explore the function of linear segments of these enzymes, a series of 14 EPT1-CPT1 chimeric gene constructs and the parental wild-type genes were expressed in a cpt1 ept1 double null mutant background completely devoid of phosphoamino alcohol transferase activity. Eleven of the chimeric genes expressed functional enzymes. The CDP-amino alcohol and sn-1,2-diacylglycerol (DAG) substrate specificities and essential phospholipid cofactor requirements of the parental and chimeric enzymes were investigated using a mixed micellar assay system. Chimeric enzymes exhibited a pattern of CDP-amino alcohol affinities that defined a structural domain sufficient to confer CDP-amino alcohol specificity. When wild-type enzymes were investigated using a chemically defined series of DAGs, each possessed a distinct characteristic pattern of utilization. Chimeric enzymes exhibited DAG acyl chain specificity profiles that either conformed to parental wild-type patterns or represented novel substrate specificities. Correlation of these outcomes with their underlying structural modifications permitted the assignment of an internal, linear region of 218 amino acids sufficient to confer DAG acyl chain specificity; this region contained three predicted transmembrane segments. Neither wild-type enzyme showed significant acyl chain selectivity with respect to phospholipid activation when a homologous series of chemically defined phosphatidylcholines were employed, suggesting that enzyme recognition of the fatty acyl moieties of the DAG substrate and phospholipid activator is fundamentally different. Analysis of chimeric enzymes dependence on phospholipid activators suggested the involvement of discontinuous protein segments participating in the interaction with phospholipid cofactors.