Since diseases of the neural retina and optic nerve can result in alteration of biological membranes, this study determines similarities and differences in the membrane phospholipid content of the neural retina, optic nerve head, and optic nerve to serve as baseline data. Neural retina, optic nerve head, and optic nerve were dissected, isolated as 5 sets from 20 rabbits and frozen in liquid N2. Separate pooled-tissue extracts were prepared for each set of tissues and phosphorus-31 nuclear magnetic resonance (31P NMR) analyses performed. Ten phospholipids were quantified (respective neural retina, optic nerve head, and optic nerve mole % are given for the 5 major phospholipids detected): phosphatidylcholine (PC), 44.61, 27.67, 26.40; PC plasmalogen or alkylacyl PC (CPLIP); phosphatidylinositol (PI); sphingomyelin (SM); phosphatidylserine (PS), 12.63, 14.77, 15.09; phosphatidylethanolamine (PE), 21.21, 9.59, 8.69; PE plasmalogen (EPLAS), 11.07, 30.96, 33.93; an unidentified (unknown) phospholipid (U) at the chemical-shift value of 0.13 ppm; diphosphatidylglycerol (DPG); and phosphatidic acid (PA), 0.46, 2.92, 1.57. Significant differences between the various tissues were determined by the one-way analysis of variance, using a Scheffé range value of P < 0.05. The neural retina in all phospholipids detected except for the uncharacterized (unknown) phospholipid was significantly different from the optic nerve head tissue. The optic nerve head was significantly different from the optic nerve in PC, CPLIP, PE, EPLAS, U, DPG, and PA. The data provide a baseline for studies on pathologically changed neural retina, optic nerve head, and optic nerve.