The alk-1-enyl bond in plasmenylethanolamine is formed from plasmanylethanolamine by the action of a microsomal cytochrome b5-dependent desaturase. However, the origin of the alk-1-enyl linkage in plasmenylcholine, a significant subclass of phospholipids in heart tissues of certain animal species, is not yet known. We have used neonatal rat myocytes as a model to study the biosynthesis of plasmenylcholine in the present studies since they have a phospholipid composition and subclasses of 1,2-diradyl-sn-glycero-3-phosphocholine (-GPC) similar to those of neonatal rat hearts. When equal concentrations of [3H]hexadecyllyso-GPC or [3H]hexadecyllyso-sn-glycero-3-phosphoethanolamine (-GPE) are incubated under identical conditions with myocytes for 4, 12, and 24 h, the rate of plasmenylcholine formation is faster from [3H]hexadecyllyso-GPE than from [3H]hexadecyllyso-GPC. Also, when [3H]alkyllyso-GPC and alkyllyso-[N-methyl-14C]GPC are incubated with rat myocytes for various times up to 24 h, the 3H/14C ratio in the diacyl-GPC plus alkylacyl-GPC fraction and alkyllyso-GPC remains relatively constant (3H/14C = 2.7), whereas the 3H/14C of plasmenylcholine increases from 0.3 at 2 h to 1.7 after 24 h. Finally, when the rat myocytes are prelabeled with [3H]alkyllyso-GPE for 4 h and then reincubated with either [14C]choline or [14C]methionine for 1 or 3 h, both [14C]choline and [14C]methionine are incorporated into plasmenylcholine, except the 14C/3H is much higher (5- to 15-fold) in the [14C]choline-labeled plasmenylcholine than in the [14C]methionine-labeled plasmenylcholine. Collectively, our data show plasmenylcholine is not directly derived from plasmanylcholine or lysoplasmanylcholine, but instead is formed from plasmenylethanolamine via some type of hydrolytic exchange mechanism, and the contribution of plasmenylethanolamine through methylation to the synthesis of plasmenylcholine is of limited capacity.