Selected neurons in the cortex and other regions of the brain of adult mice stain with Vicia villosa agglutinin (VVA) conjugated to horseradish peroxidase (HRP). This staining demonstrates glycoconjugate with terminal N-acetylgalactosamine (GalNAc) in periodic foci on the neuronal surface. However, brains from mice at 1 week of age lacked neurons with surface reactivity for VVA-HRP but showed neurons with faint cytoplasmic staining. Two weeks postnatally, infrequent cortical neurons located mostly in layer IV disclosed light surface affinity for VVA-HRP. The deep cerebellar nucleus at 2 weeks revealed VVA-positive neurons that were less abundant than in adults but exceeded in number the stained neurons in the cortex of 2-week-old mice. By 3 weeks of age the prevalence of positive neurons in cortical layers II-VI and the deep cerebellar nucleus and the intensity of the surface staining approximated that observed in adult mice. No staining of fiber tracts was observed 1 week postnatally. However, certain tracts exemplified by decussating fibers in the basal pontine region showed strong VVA-HRP reactivity in brains of 2-week-old mice as in adults. Acquisition of VVA surface affinity 3 weeks postnatally coincided with the appearance of cytosolic immunostaining for glutamic acid decarboxylase in a population of GABAergic neurons in the cortex. These findings demonstrate the postnatal development of GalNAc-containing glycoconjugate associated with selected neurons and the variable acquisition of the glycoconjugate among these neurons possibly related to onset of function.