The clinical, pathological and biochemical features of a neuroaxonal dystrophy resulting from the deficient activity of lysosomal alpha-N-acetylgalactosaminidase are described. This neurodegenerative disorder was recognized in two brothers who had the typical clinical manifestations and neuropathological lesions observed in patients with Seitelberger disease, the infantile form of neuroaxonal dystrophy. Axonal 'spheroids' were observed histologically in the grey matter, and ultrastructural examination revealed the characteristic formations in dystrophic axons in the myenteric plexus and neocortex. Using a newly synthesized fluorogenic substrate, 4-methylumbelliferyl-alpha-N-acetylgalactosaminide, the markedly deficient activity of alpha-N-acetylgalactosaminidase was demonstrated in the affected brothers while their consanguineous parents had intermediate activities, consistent with the autosomal recessive transmission of this disease. No detectable alpha-N-acetylgalactosaminidase was seen in immunoblots using monospecific rabbit antihuman alpha-N-acetylgalactosaminidase antibodies. Abnormally increased amounts of urinary glycopeptides were observed by high resolution thin layer chromatography. Analytical studies identified four of the accumulating urinary compounds, the blood group A trisaccharide GalNAc alpha 1----3(Fuc alpha 1----2)Gal and three O-linked glycopeptides, GalNAc alpha 1----O-serine and -threonine, NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----6)GalNAc alpha 1----O-serine and -threonine, and NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6(NeuNAc alpha 2----3Gal beta 1----3)GalNAc alpha 1----O-serine and -threonine. Of eight unrelated patients diagnosed as having infantile neuroaxonal dystrophy by pathological studies, none had deficient alpha-N-acetylgalactosaminidase activity, emphasizing the biochemical heterogeneity underlying this diagnostic entity. These findings document the first delineation of a metabolic defect in an inherited neuroaxonal dystrophy and suggest that the axonal pathology in this disorder, and perhaps in the other neuroaxonal dystrophies, results from abnormal glycoprotein metabolism involving O-linked glycopeptides.