Quantal release of the principal excitatory neurotransmitter glutamate requires a mechanism for its transport into secretory vesicles. Within the brain, the complementary expression of vesicular glutamate transporters (VGLUTs) 1 and 2 accounts for the release of glutamate by all known excitatory neurons. We now report the identification of VGLUT3 and its expression by many cells generally considered to release a classical transmitter with properties very different from glutamate. Remarkably, subpopulations of inhibitory neurons as well as cholinergic interneurons, monoamine neurons, and glia express VGLUT3. The dendritic expression of VGLUT3 by particular neurons also indicates the potential for retrograde synaptic signaling. The distribution and subcellular location of VGLUT3 thus suggest novel modes of signaling by glutamate.