Vesicular glutamate transporter 3 (VGLUT3) expression has been specifically localized to brain regions rich in serotonergic cells. It has been suggested that this transporter may contribute to the regulation of extracellular glutamate concentrations via a nonsynaptic mechanism. In this study, we examine the colocalization of vesicular glutamate transporter 3 immunoreactivity with serotonin immunoreactivity in the dorsal and median raphe nuclei of Syrian hamsters. Brain sections from adult hamsters were fluorescently labeled for serotonin-ir and VGLUT3-ir and examined using confocal microscopy. The results indicate that most serotonergic cells of the midbrain raphe also expressed vesicular glutamate transporter 3. In addition, nonserotonergic cells in these brain regions also show immunoreactivity for the transporter. These data confirm previous findings of vesicular glutamate transporter 3 expression in serotonergic and nonserotonergic neurons in rats. These findings suggest that the location of vesicular glutamate transporter 3 may be as much a function of neuroanatomical location as of the neurochemical identity of the expressing neurons.