The principle neuronal output of the neostriatum comes from medium spiny neurons that project from the caudate/putamen to the globus pallidus and substantia nigra. Although current evidence generally indicates that γ‐aminobutyric acid (GABA) is the principal neurotransmitter in this pathway, this cannot account for the excitatory synaptic activity present among cultures of striatal neurons or the short latency excitatory postsynaptic potentials which often proceed or obscure inhibitory activity evoked by striatal stimulation. In this study, retrograde transport of [3H]D‐aspartate has been used to demonstrate striatopallidal and striato‐nigral neurons that possess a high‐affinity uptake system for glutamate and aspartate and are therefore putatively glutamatergic. Injections of [3H]D‐aspartate into the globus pallidus or substantia nigra, pars reticularis of the rat retrogradely labeled mediumsized neurons throughout the rostral‐caudal extent of the neostriatum. To characterize this population further, adjacent sections were immunoreacted with antibodies to either GABA, glutamic acid decarboxylase (GAD), calbindin, or parvalbumin prior to autoradiographic processing. Under these conditions, autoradiographically labeled neurons displayed positive immunoreactivity for GABA, GAD, or calbindin. Autoradiographic label did not colocalize with parvalbumin immunoreactivity. The colocalization of anatomical markers of GABAergic and glutamatergic neurotransmission raises the possibility that both neurotransmitters are functionally expressed within single striatal projection neurons. © 1994 Wiley‐Liss, Inc.