Two proteins previously known as Na(+)-dependent phosphate transporters have been identified recently as vesicular glutamate transporters (VGLUT1 and VGLUT2). Together, VGLUT1 and VGLUT2 are operating at most central glutamatergic synapses. In this study, we characterized a third vesicular glutamate transporter (VGLUT3), highly homologous to VGLUT1 and VGLUT2. Vesicles isolated from endocrine cells expressing recombinant VGLUT3 accumulated l-glutamate with bioenergetic and pharmacological characteristics similar, but not identical, to those displayed by the type-1 and type-2 isoforms. Interestingly, the distribution of VGLUT3 mRNA was restricted to a small number of neurons scattered in the striatum, hippocampus, cerebral cortex, and raphe nuclei, in contrast to VGLUT1 and VGLUT2 transcripts, which are massively expressed in cortical and deep structures of the brain, respectively. At the ultrastructural level, VGLUT3 immunoreactivity was concentrated over synaptic vesicle clusters present in nerve endings forming asymmetrical as well as symmetrical synapses. Finally, VGLUT3-positive neurons of the striatum and raphe nuclei were shown to coexpress acetylcholine and serotonin transporters, respectively. Our study reveals a novel class of glutamatergic nerve terminals and suggests that cholinergic striatal interneurons and serotoninergic neurons from the brainstem may store and release glutamate.