The specificity of phosphatidylethanolamine (PE) N-methyltransferase for molecular species of PE has been investigated. Phosphatidylcholine (PC), synthesized by incubation of [methyl-3H]S-adenosyl-L-methionine with microsomes or pure enzyme (Ridgway, N. D., and Vance, D. E. (1987) J. Biol. Chem. 262, 17231-17239) plus microsomal PE, had a distribution of methyl label in molecular species similar to the mole percent distribution of molecular species in the precursor PE. A similar lack of specificity was observed with PE that was synthesized from egg PC by transphosphatidylation with phospholipase D. Phosphatidyl-N-monomethylethanolamine (PMME) and phosphatidyl-N,N-dimethylethanolamine (PDME), both with the acyl composition of egg PC, were methylated by the pure enzyme and showed a distribution of labeled molecular species in PDME and PC, respectively, similar to the mole percent distribution of egg PC. Results with synthetic PEs and pure methyltransferase showed higher rates of methylation with more unsaturated species. Long chain saturated PEs (e.g. dipalmitoyl-PE) were not methylated by the enzyme. Maximal methylation rates were obtained with two or more double bonds in the substrate PE. Rates of methylation of the saturated and monoenoic PEs could be enhanced when 40 mol % polyunsaturated-rich microsomal PC was included in the mixed micelles. PC isolated from primary cultures of rat hepatocytes pulsed with [methyl-3H]methionine was analyzed by high performance liquid chromatography. Initially, the labeling pattern of PC molecular species varied slightly from that of total hepatocyte PE and hepatocyte microsomal PE. 1-Palmitoyl-2-docosahexaenoyl-PC had the highest specific activity at the end of the pulse and was preferentially labeled relative to the mole percent distribution of hepatocyte PE molecular species. During the 24-h chase period both the percent distribution of label and specific activity of this species of PC declined. In the same time period, there was a corresponding increase in specific activity and percent distribution of label in 1-palmitoyl and 1-stearoyl species with linoleate and arachidonate in the sn-2 position.