Essential fatty acid (EFA) deficiency was induced by feeding pregnant rats a fat-free diet 10--12 days after impregnation and maintaining the offspring on this diet until 120 days of age. EFA-deficiency rats demonstrated marked alterations in the fatty acid composition of ethanolamine phosphoglycerides (EPG's) from myelin subfractions. A decrease in the fatty acids of the linoleic (n-6) and linolenic (n-3) families was accompanied by an increase in the non-essential fatty acids of the oleic (n-9) family. These alterations decreased the unsaturation index of heavy myelin by 23% and that of light myelin by 10%. The EPG fatty acid composition of heavy myelin from control animals contained a higher percentage of polyunsaturated fatty acids than the light myelin which contained more monounsaturated fatty acids. These differences may be a reflection of distinct anatomical locations or functional properties of the subfractions. The differences between light and heavy myelin EPG fatty acids were not maintained during EFA deficiency. Morphologically, 1 mum thick sections revealed vacuoles within the optic nerve of EFA-deficient rats. Ultrastructurally these vacuoles were identified as fibers undergoing Wallerian degeneration and fibers demonstrating intramyelinic splitting. No qualitative changes were found in oligodendrocytes, astrocytes or vascular elements within EFA-deficient optic nerve. EFA deficiency did not alter the diameter of fibers within the optic nerve. These results show that although there is no apparent decrease in the degree of myelination within the optic nerve, morphological changes do occur in fibers of EFA-deficient optic nerve concomitantly with alterations in the EPG fatty acids of myelin subfractions.