In Dictyostelium discoideum, (N-acyl)ethanolamine glycerophospholipids disappear as the amoebae aggregate, whereas the amount of ethanolamine glycerophospholipids remains relatively constant, suggesting that each type of ethanolamine-containing phospholipid might have a separate metabolic pathway. To study their metabolism, phosphatidylethanolamine and phosphatidyl(N-acyl)ethanolamine containing either [14C]ethanolamine or a 14C-labeled sn-2 fatty acyl group were incubated with D. discoideum homogenates, and the conversion of the substrates into radioactive products was monitored. At pH values 3.8 and 4.5, phosphatidyl(N-acyl)ethanolamine was hydrolyzed by a phospholipase A1 to form the sn-2 acyl form of the lipid. Only minor hydrolysis occurred at pH values of 5.2 or higher. (N-acyl)Ethanolamine was also released by a phospholipase D type activity at 0.1 the rate of the lysophospholipid formation. Phosphatidyl(N-acyl)ethanolamine was not hydrolyzed to form phosphatidylethanolamine or water soluble components. At pH 7.2 and at the low pH range of 3.8-4.5, phosphatidylethanolamine was hydrolyzed to lysophosphatidylethanolamine, which was then further degraded to water-soluble components. At pH 7.2, a phospholipase A2 initially hydrolyzed the phosphatidylethanolamine, whereas at the low pH range a phospholipase A1 was the most active enzyme. Although both types of ethanolamine-containing phospholipid were hydrolyzed by a phospholipase A1 at the low pH range, phosphatidylethanolamine hydrolysis was more sensitive to inhibition by Trition X-100, and phosphatidylethanolamine was hydrolyzed to water-soluble components, whereas phosphatidyl(N-acyl)ethanolamine was not. At pH 7.2, phosphatidylethanolamine was hydrolyzed, but phosphatidyl(N-acyl)ethanolamine was not hydrolyzed at all. These results indicate that there are separate routes of degradation for the two types of ethanolamine-containing phospholipid in D. discoideum.