Immunohistochemical localization of ganglioside GM1 using 3 monoclonal antibodies (C3 and D3 reacting exclusively with GM1 and C4h2 reacting also with other gangliosides) showed different staining patterns in rat brain regions (cerebellum, cerebral cortex and hippocampus). Staining in all brain areas was punctate and appeared to be restricted to surfaces of cells and their processes. In spite of similar reactivity to GM1, C3 and D3 showed qualitatively and quantitatively different and highly selective localization in all regions with no staining in white matter. In cerebellum, staining with C3 was predominantly associated with granular layer; staining with D3 was limited to Purkinje cell surfaces and surrounding structures. In cortex, staining with C3 was seen outlining large pyramidal neurons and fibers in cross-section, whereas sites stained with D3 appeared to be fewer, smaller and differed in location. In hippocampus, staining patterns were similar with both C3 and D3, outlining large pyramidal neurons of Ammon's horn and granular cells of dentate gyrus as well as glomerular structures. Staining with C4h2 was topographically similar, but over 10-fold more extensive and was present in white matter.