The effects of choline, ethanolamine and its N-methyl analogs, different fatty acids, and L-methionine on phospholipid biosynthesis via the CDP-ester pathways and the methylation pathway were studied in rat hepatocytes. Phosphatidylethanolamine synthesis was stimulated severalfold by 0.02 to 0.1 mM ethanolamine, especially in the presence of long chain unsaturated fatty acids. At higher concentrations of ethanolamine, phosphorylethanolamine accumulated but the level of CDP-ethanolamine and the rate of phosphatidylethanolamine synthesis did not increase further. The rate of phosphatidylcholine synthesis via the CDP-ester pathway responded in a way analogous to that of phosphatidylethanolamine synthesis upon the addition of choline and fatty acid, except that a 10- to 20-fold higher concentration of choline was required for maximal stimulation, probably due to the rapid oxidation of choline to betaine. Phospholipids containing N-monomethyl- or N,N-dimethylethanolamine were efficiently formed from the corresponding free bases in the absence of ethanolamine and choline. Ethanolamine, but not other bases, inhibited completely phospholipid formation from N-monomethylethanolamine, probably as a result of competition at the level of CDP-ester formation. The data indicate that the cytidylytransferase reactions are rate-limiting steps in the synthesis of phosphatidylethanolamine and probably also phosphatidylcholine. In addition, the availability of diacylglycerol and its fatty acid composition may significantly affect the rate of phospholipid synthesis. The rate of phosphatidylcholine formation via phospholipid N-methylation approximately doubled when L-methionine was added at concentrations similar to that in rat plasma. Under these conditions the rate of phosphatidylcholine synthesis via this pathway was 20 to 40 percent of that via diacylglycerols and CDP-choline. The methylation of phosphatidylethanolamine to phosphatidylcholine remained essentially constant when the rate of phosphatidylethanolamine synthesis was varied 8-fold, but was significantly reduced when the formation of N-monomethyl- or N,N-dimethylphospholipid was stimulated by addition of the corresponding base. These phospholipids not only replaced phosphatidylethanolamine as the substrate for methylation but also increased the rate of phosphatidylcholine formation via this pathway. A method for the determination of nanomole amounts of different ethanolamine compounds is described.