A combination of retrograde tracers and immunostaining was employed to test whether corticospinal tract (CST) neurons in rats may use amino acid excitatory neurotransmitters. CST neurons were identified following injections of either Diamidino Yellow (DY) or colloidal gold-labeled enzymatically inactive horseradish peroxidase conjugated to wheat germ agglutinin (WGAapoHRP-Au) in the spinal cord. As retrograde tracers, the two substances seemed to be equally effective, but WGAapoHRP-Au was better suited than DY as a tracer to use in combination with immunocytochemistry. Sections through the primary sensorimotor cortex, which contained the bulk of identified CST neurons, and the secondary somatosensory cortex were processed with antisera raised in rabbits against glutamate (Glu) or aspartate (Asp) conjugated by glutaraldehyde to hemocyanin. In rats with DY injections, about 60-75% of the CST neurons were Glu-immunopositive, with higher ratios in SI and MI than in SII. Similar results were obtained in all areas examined from the rats with injections of WGAapoHRP-Au. Only sections from rats with injections of WGAapoHRP-Au were processed for Asp immunostaining. In this material, between 65 and 75% of the CST neurons were Asp-immunopositive, with a slightly higher ratio in SI and MI than in SII. The possibility that these results might reflect limited penetration of the antiserum and/or staining of the same population of CST neurons by either antiserum was addressed in sections processed with both the Glu and Asp antisera. In sections incubated in a mixture of the two antisera, the percentage of immunostained CST neurons was higher, about 90%, than in sections processed for only one of the two antisera. Furthermore, in rats in which Glu and Asp antibodies were visualized by two distinguishable immunostainings, four populations of CST neurons were identifiable: 1) neurons only immunopositive for Glu, 2) neurons only immunopositive for Asp, 3) neurons likely to be stained by both, and 4) neurons immunonegative for both antisera. Twenty-five to 30% of CST neurons were positive for only one antiserum, and about 50% were positive for both. No preferential distribution was evident for any one of these populations of neurons. However, perikaryal cross-sectional areas were larger for the double-stained than for the single-stained CST neurons. Glutamergic and aspartergic transmission in CST neurons has been proposed in several publications in which methods other than immunocytochemistry were employed.(ABSTRACT TRUNCATED AT 400 WORDS)