GM1 ganglioside is a receptor for the B subunit of cholera toxin. In lymphocytes, B subunit elicits an influx of extracellular Ca++ (Dixon et al., 1987). To investigate this signaling pathway in glia, we assessed the presence of GM1 ganglioside on the surface of cultured murine central nervous system (CNS) glia by binding of fluorescein-labeled B subunit. B subunit binding was compared to binding of peanut agglutinin, wheat germ agglutinin, and Bandeiraea (Griffonia) simplicifolia lectin (BSL)I, a microglial marker. Antibodies to glial fibrillary acidic protein, A007/O4 antigens, and galactocerebroside were used to identify astrocytes, immature oligodendrocytes (OLs) and mature OLs, respectively. Binding patterns differed based on cell type and developmental stage. Wheat germ and peanut agglutinins bound to the surface of microglia, astrocytes, and immature OLs; neither lectin bound to any significant extent to the surface of membrane sheets of mature OLs, although wheat germ agglutinin was rapidly endocytosed. Cells identified as microglia by BSL I binding and morphology were the only cells to stain brightly on the surface with B subunit. Thus, surface GM1 ganglioside appears to be a highly enriched marker for microglia in these mixed glial cultures. The effects of B subunit on intracellular Ca++ were examined by laser cytometry in glial cultures loaded with Indo-1. No Ca++ responses were observed in microglia. Mature OLs were examined for Ca++ responses to B subunit before and after surface levels of GM1 ganglioside were increased by incubation with exogenous GM1 ganglioside. Again, no Ca++ responses were observed. Thus, cultured microglia and mature OLs do not have the GM1-mediated signal transduction pathway seen in lymphocytes. However, the presence of GM1 ganglioside on microglia may play a role in giving rise to antibodies to this glycolipid in some CNS inflammatory diseases.