Erucic acid (delta 13-docosenoic acid), labeled with 14C in the 1- or 14-position, was incorporated into fetal calf serum and fed to beating, neonatal rat myocardial cell in culture. Uptake of the docosenoic acid during the first 6 hr of incubation was 41 nM/hr/mg protein in 7-day old cells and 29 nM/hr/mg protein in 14-day old cells. Fifty-seven percent of the 14C-activity was taken up from the medium in 24 hr, of which 77% was in the cells and 23% was unaccounted for. Of the 14C-activity taken up, 26% was in extractable lipid, with two-thirds in neutral lipid and one-third in phospholipid. Within the neutral lipid fraction, 88% of the 14C-activity was present in triglycerides; while in phospholipids, 66% of the 14C-activity was in phosphatidylcholine (PC); 14% in phosphatidylethanolamine (PE); 6% in sphinogomyelin (SPH) and 1% or less in cardiolipin (DPG). PC had the highest specific activity, followed by SPH and PE. The specific activity of PE was one-half that of SPH when the 14C-erucic acid substrate was labeled at the carboxyl position, but increased to equal that of SPH when the substrate was labeled at the double bond. The fatty acids of PC, PE, and SPH were influenced by erucic acid in the growth medium, but the amounts of each phospholipid were not affected. It is proposed that the altered fatty acid composition associated with incorporation of erucic acid or its metabolites into PC, PE, and SPH may affect integrity and function of heart cell membranes.