Vesicular glutamate transporter (VGLUT) plays an essential role in L-glutamate signaling in neurons and some peripheral tissues through vesicular storage of L-glutamate in secretory vesicles. To investigate the topology of VGLUT in membranes, we prepared site-directed antibodies against the amino-terminal (anti-N), 1st putative loop (anti-L), and carboxyl terminal (anti-C) regions. None of the antibodies reacted with VGLUT2 expressed in COS cells because they could not gain access to the antigen. However, both the anti-N and anti-C antibodies recognized VGLUT2 when the cells were permeabilized with digitonin, while the anti-L antibodies did not. Immunological reactivity to anti-L-antibodies appeared when the cells were permeabilized with Triton X-100. These results suggest that both the amino-terminal and carboxyl-terminal regions of VGLUT2 in membranes face the cytoplasm while the 1st loop faces the lumen.