Freshly isolated rat hepatocytes were incubated for 20 and 60 min with [U-14C]glycerol and unlabeled palmitic (16:0), oleic (18:1), or arachidonic (20:4) acid, added as albumin complex in 10% ethanol. Each fatty acid increased glycerol incorporation into total lipids by a factor of 8-10 over control, whereas ethanol alone (final concentration 100 mM) yielded a threefold increase of glycerol uptake. Glycerol incorporation stopped after 20 min and cellular acyl turnover continued in the absence of useable labeled substrate. In each case, radioactivity recovered in hepatocyte lipids was present primarily in triacylglycerol (37-64%), phosphatidylcholine (22-37%), and phosphatidylethanolamine (10-22%). Separation by high-performance liquid chromatography of the diacylglycerol dinitrobenzoates derived from phosphatidylcholine showed that the molecular species had drastically different labeling patterns in the presence of the exogenous fatty acids, whereas the pattern obtained in the presence of ethanol alone was virtually the same as that for the control incubations. The labeling patterns indicated that exogenous fatty acids, including arachidonic acid, were incorporated into phosphatidylcholine primarily by the de novo pathway yielding highly labeled species with the exogenous fatty acid esterified at both the sn-1 and sn-2 positions of glycerol. After 20 min incubation with arachidonic acid, the 20:4-20:4 phosphatidylcholine contained about one-half of the [U-14C]glycerol label recovered in this lipid class. The data also showed that newly synthesized molecular species were extensively remodeled within 1 h.