Immunofluorescence analysis was used to study the cellular localization of glucose transporters 1 and 3 (GLUT1 and GLUT3) in primary rat neuronal and glial cultures. In primary cultured cerebellar granule neurons and cortical neurons, GLUT3 was detected in a pattern consistent with a generalized cell surface distribution. GLUT3 distribution corresponded most closely with the neural cell adhesion molecule (NCAM), and showed overlapping but distinct distributions compared to synaptophysin, microtubule-associated protein 2 (MAP2), neurofilament protein, and growth-associated protein (GAP43). Culture of neurons in the presence of glia did not alter the cellular localization of GLUT3. GLUT1 was detectable in primary cerebellar granule neurons both at the cell surface and in the cytoplasm, and appeared decreased in neurons cocultured with glia. GLUT1, but not GLUT3, was detected in glial fibrillary acidic protein (GFAP)-positive astrocytes present in mixed neuronal-glial cultures derived from cerebellum and cerebral cortex, as well as in cortical astrocyte cultures. GLUT1, but not GLUT3, was also detected in microglia and oligodendrocytes present in these cultures. This study indicates a generalized cell surface expression of the glucose transporters expressed in neurons and glia, rather than selective targeting to different cellular domains or subcellular locations.