The 2-ester bond of 14C-2-arachidonyl phosphatidylethanolamine (PE) was hydrolyzed faster than that of 3H-2-arachidonyl phosphatidylcholine (PC) by human pancreatic phospholipase A2 (PLA2) with mixed PE-PC (1:9 w/w) liposomes of pure sonicated PE or PC as substrate. The PC portion of the mixed PE-PC liposomes was more readily attacked by PLA2 than the PC of pure PC liposomes. At different bile salt concentrations (sodium taurocholate (NaTC), 0-3 mM, and sodium taurodeoxycholate (NaTDC), 0-4 mM) the rates of hydrolysis varied with similar patterns for both phospholipids of the mixed liposomes. The rate of hydrolysis was optimal at a bile salt concentration of 0.75-1.5 mM NaTC and 1.0-2.0 mM NaTDC and decreased at higher concentrations. The pure PE substrate was efficiently hydrolyzed also without bile salts. This may have implications for the absorption of polyunsaturated phospholipid fatty acids in patients with bile salt deficiency. Separation of phospholipid classes from human bile by high-performance liquid chromatography and analysis of fatty acid composition indicated that PE contained 5.3% of the phospholipid arachidonic acid and 9.8% of the docosahexaenoic acid mass, but only 1.7% of the total phospholipid mass. Bile and dietary PE should not be overlooked as sources of arachidonic and docosahexaenoic acid for the small intestine.