Mesoaccumbens fibers are thought to co-release dopamine and glutamate. However, the mechanism of co-release of dopamine and glutamate is unclear, and the co-release by mesoaccumbens fibers has not been documented. By electron microcopy we showed that some mesoaccumbens fibers have vesicular transporters for dopamine (VMAT2) in axon-segments continuous with axon-terminals that lack VMAT2, but contain vesicular glutamate transporters type 2 (VGluT2). In vivo overexpression of VMAT2 did not change segregation of the two vesicular types, suggesting highly regulated mechanisms for maintaining this segregation. The mesoaccumbens axon terminals containing VGluT2-vesicles make asymmetric synapses; commonly associated with excitatory signaling. By optogenetics, we showed release of dopamine and glutamate from mesoaccumbens fibers. These findings reveal a complex type of signaling by mesoaccumbens fibers in which dopamine and glutamate although can be released from the same axons; they are not normally released at the same site or from the same synaptic vesicles.