We and others have previously reported that glucose transporter 1 (GLUT1)-like 45 kDa protein is localized to parenchymal cells in the brain. However, the precise cellular localization has remained unclear. In the present study, we examined the cellular localization of GLUT1 in the rat brain by double immunostaining methods and immunoelectron microscopic analysis using a rabbit antiserum specific to GLUT1. Western blot analysis of the rat brain revealed that the antiserum detected a strong band with a molecular weight of 45 kDa and a weak band of about 55 kDa, which corresponded respectively to the known molecular weights of the GLUT1 proteins in the brain parenchymal cells and the brain microvessels. Immunohistochemical staining revealed a large number of GLUT1-immunoreactive glial cells and microvessels in almost every region of the brain. Double immunofluorescence analysis demonstrated that the GLUT1-like 45 kDa protein occurred in many galactocerebroside-positive oligodendrocytes and in some glial fibrillary acidic protein (GFAP)-positive astrocytes. No GLUT1-immunoreactivity was observed in OX42-positive microglia. Immunoelectron microscopic examination confirmed that the GLUT1-immunoreactivity was mainly localized in the cytoplasm of the oligodendrocytes and astrocytes. The results indicate that the 45 kDa form of GLUT1 protein exists in the glial cells including astrocytes and oligodendrocytes.