The retrograde transport of a tracer has been combined with peroxidase immunocytochemistry to verify whether corticostriatal (CS) neurons contain in their cell bodies high levels of glutamate (Glu) and aspartate (Asp). Injections of WGAapoHRP-Au in the caudate/putamen of adult rats produced retrograde labeling of a large number of layer V neurons of wide regions of the ipsilateral cerebral cortex; fewer labeled neurons were also found on the contralateral side. In all experiments, most CS neurons were seen in the agranular frontal cortex, in both the medial and the lateral subdivisions. Moreover, numerous retrogradely labeled neurons were observed in the cingulate cortex and in the granular parietal cortex, depending on the location of the injection site in the various experiments. The majority of CS neurons examined were immunostained using antibodies against glutaraldehyde-conjugates of Glu or Asp. Glu immunopositive neurons resulted 52-61% of CS neurons. Asp immunopositive neurons ranged between 53% and 62%. In the cortical tissue where Glu and Asp antisera were visualized simultaneously, up to 96% of the CS neurons were immunostained. The latter finding indicates that the populations of Glu and Asp immunopositive neurons are largely segregated and that virtually all cortical neurons projecting to the striatum contain high concentrations of Glu and/or Asp, thus corroborating the hypothesis that CS projections use excitatory amino acids as neurotransmitters.