Using standard immunohistochemical and histochemical techniques, we have examined the neurochemical characteristics of a subpopulation of locomotor-related neurons as labeled by the activity-dependent marker c-fos. Results were compared to those obtained from a small sample of intracellularly labeled locomotor-related neurons. In the paralyzed, decerebrate cat, fictive locomotion was evoked by electrical stimulation of the mesencephalic locomotor region. Most c-fos-immunoreactive neurons were distributed in medial lamina VI and VII and in lamina VIII and X. Double labeling of c-fos with various cytochemical markers revealed that about one-third of the c-fos-immunoreactive neurons were choline acetyltransferase immunoreactive, about one-third were glutamate immunoreactive, and about one-third were aspartate immunoreactive. In addition, approximately 15% of the c-fos-labeled neurons contained NADPH-diaphrorase reaction product, while almost 40% appeared to receive close contacts from calcitonin gene-related peptide-immunoreactive fibers and boutons. Choline acetyltransferase- or aspartate immunoreactivity was observed in some intracellularly labeled neurons. These findings have implications regarding the putative neurotransmitters utilized by subpopulations of locomotor-related neurons in the cat spinal cord.