Biochemical analyses were performed on cerebral biopsies from three siblings with metachromatic leukodystrophy, and from autopsy tissue obtained 6 months later. The lipids of all gray matter samples were relatively normal, with the exception of an elevated sulfatide: cerebroside ratio. The white matter samples were grossly abnormal and showed a progression in severity of biochemical abnormalities with duration of the disease. Sulfatides were 4-8-fold higher than normal, and the ratio of cerebrosides to sulfatides ranged from 0.13 to 0.26, compared to the normal value of about 4.0. Myelin was isolated in very low yield, but had normal morphology. As others have reported, it had the same chemical defect as whole white matter; sulfatides were 7-8 times higher than normal and cerebrosides were reduced by more than half. The fatty acid compositions of the myelin sphingolipids were found to be of much longer average chain length than those in affected white matter. In myelin, the unsubstituted fatty acids of cerebrosides and sphingomyelin had a higher percentage of short chains than found in normal myelin, but were less abnormal than those in whole white matter, whereas the unsubstituted and alpha-hydroxy acids of sulfatides and the alpha-hydroxy acids of cerebrosides were not deficient in long chains. These data indicate that there are at least two compartments of sphingolipids in MLD white matter, each having different average chain lengths.