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Phospholipids of optic nerve (n = 30) from 5.6-kg rabbits were analyzed by ³¹P nuclear magnetic resonance (NMR) spectroscopy. Phospholipid metabolites detected were as follows (mol %): phosphatidylcholine (PC; 25.82 ± 0.12), PCplasmalogen/ alkylacyl PC (2.07 ± 0.13), sphingosylphosphorylcholme (1.12 ± 0.20), phosphatidylinositol (PI; 2.17 ± 0.21), lysoPC (0.85 ± 0.06), sphingomyelin (12.52 ± 0.10); phosphatidyl-serine(PS; 14.38 ± 0.11), phosphatidylethanolamine(8.98 ± 0.11), ethanolamine plasmalogen (28.99 ± 0.30), unidentified phospholipid (1.10 ± 0.01), phosphatidic acid (PA; 1.72 ± 0.06), and lyso PS (0.28 ± 0.10). The bulk of the ethanolamine phosphatide is in the form of its plasmalogen, which is the major phospholipid detected. The choline plasmalogen, or a reduced derivative thereof, also is present; thus, a significant phospholipid biosynthetic pathway for optic nerve tissue involves the plasmalogen route, which is a pathway distinct from the PA route responsible for the synthesis of PS, PI, and PC. This new ³¹P NMR lipid analytical technique offers potential for studying optic nerve phospholipid metabolism and degenerating optic nerve tissue, since the technique can accurately quantitate (1) both plasmalogen and nonplasmalogen phospholipids, (2) minor phospholipid components, and (3) previously undetected phospholipids.