The serotonergic dorsal raphe nucleus (DR) is an area enriched with cell bodies and axons containing the third vesicular glutamate transporter, VGLUT3. However, the role of VGLUT3-containing axons in modulating activity of serotonin (5-HT) neurons within the DR remains poorly understood. In this study, neurochemical features and topography of VGLUT3-containing cell bodies and axons in the DR were examined. Since many 5-HT cells have been reported to express VGLUT3, the distribution of dually labeled axons was examined within the DR. Axons containing both VGLUT3 and 5-HT immunolabeling had a topographic distribution: they innervated the ependyma and ramified within the caudal DR at the base of the aqueduct, an area known to give rise to ependymal innervation. Thus VGLUT3 is only present in a specific subcomponent of recurrent 5-HT axon collaterals. Remaining VGLUT3 axons were only rarely dually immunolabeled for markers of monoamines, GABA, or acetycholine, suggesting these axons contain a predominance of glutamate-filled vesicles. Since the substance P receptor, neurokinin 1 (NK1), has previously been associated with local glutamate neurons in the DR, the relationship between NK1, 5-HT and VGLUT3 cells was examined using triple-immunolabeling. Results indicate that the majority of non-5-HT VGLUT3-containing cell bodies in the DR contain NK1 immunolabeling. Taken together, these findings indicate locally collateralizing glutamate neurons responsive to substance P contain VGLUT3.