The molecular species composition of glycerophospholipid classes in nerves of normal and experimentally diabetic rats was determined. sn-1,2-Diacylglycerol (DAG) moieties of purified phospholipids were liberated enzymatically and analyzed as the benzoate derivatives by high-performance liquid chromatography. The most abundant molecular species in phosphatidylinositol (PI) from normal nerve were 18:0/20:4 (54%) and 16:0/18:1 (17%), whereas in phosphatidylcholine (PC), 16:0/18:1 (52%), 16:0/16:0 (12%) and 18:0/18:1 (11%) predominated. In phosphatidylethanolamine and ethanolamine plasmalogen, 18:1/18:1, 16:0/18:1 and 18:0/18:1 comprised more than 60% and 75% of the molecular species, respectively. Phosphatidylserine was characterized by a high content of 18:0/18:1 (38%) and a relative abundance of the 18:1/20:0, 18:1/22:0 and 18:1/24:0 molecular species, which together accounted for over 30% of the total. The molecular species profile of phosphatidic acid did not closely resemble that of any other phospholipids or DAG. In diabetic nerve, the molecular species composition of all diacylphospholipids showed a significant decline in the content of one or more arachidonoyl-containing molecular species. The largest decline occurred in PC and the least in PI. Except in PC, 16:0/20:4 was more depressed than 18:0/20:4. In combination with previous analyses of DAG molecular species which showed a similar decline in the content of arachidonoyl-containing molecular species in nerve from experimentally diabetic rats (Zhu, X. and Eichberg, J. (1990) J. Neurochem. 55, 1087-1090), the results suggest that nerve DAG arises largely, but not entirely, from phosphoinositides and that PC could be a significant precursor, especially in diabetic nerve.