Two glucose transporter (GLUT) isoforms have been identified in brain. The GLUT1 isoform is abundant in cerebral microvessels and may be present in glia and neurons, whereas GLUT3 is probably the major neuronal glucose transporter. This study investigates whether GLUT3 is also present in microvessels from rat, human, and canine brain, by means of antisera directed against the divergent C-terminal sequences of mouse and human GLUT3. GLUT1 was detected in whole brain as two molecular mass forms: 55 kDa in microvessels and 45 kDa in cortical neuronal/glial membranes. With the aid of the appropriate antisera to the species-specific sequences, GLUT3 was detected in rat and human cortical membranes but not in isolated rat or human microvessels. These antisera failed to detect GLUT3 in either canine cortical membranes or canine microvessels, implying additional species specificity in the C-terminal sequence.