We have measured the hydrolysis of tri-, di- and monoacylglycerols and of phosphatidylcholine in the lung and have quantitated the incorporation of glyceride and phospholipid fatty acids into lung lipids. Lipolytic activity was studied in the isolated, ventilated, rat lung perfused for 100 min in a recycling system with Krebs-Ringer bicarbonate buffer containing 3% bovine serum albumin, 5.6 mM glucose, and the lipid emulsions. Triacylglycerols were hydrolyzed at significantly (P less than 0.05) lower rates than partial acylglycerols. Hydrolysis of di- and triacylglycerols was significantly lower in lungs of animals given i.v. heparin 60 min prior to death. The incorporation of fatty acids released from acylglycerols was greater into lung neutral fats than into lung phospholipids. Phosphatidylcholine was also hydrolyzed in the isolated perfused lung. The data show that (a) neutral glycerides and phospholipids are hydrolyzed in the isolated, ventilated, perfused rat lung; (b) the hydrolysis of tri- and diacylglycerols is markedly inhibited in lungs isolated from rats given heparin (100 U/kg) 1 h prior to death, whereas the hydrolysis of monoacylglycerol is unaffected by heparin administration, suggesting that tri- and diacylglycerols are hydrolyzed by endothelial lipoprotein lipase, whereas hydrolysis of monoacylglycerol is catalyzed by a separate monoacylglycerol lipase; (c) free fatty acids released by lipoprotein lipase are used by the lung for metabolic needs.