We have evaluated the pattern of c-fos expression induced in the rat spinal cord, caudal brainstem and cerebellum by a behavior that is associated with non-noxious inputs transmitted over large-diameter primary afferent fibers, namely walking for 1 h on a rotating rod. Walking on the rotating rod induced a large increase in the number of Fos-like immunoreactive neurons in regions of the cervical and lumbar spinal cord gray matter that contain neurons that respond to non-noxious stimuli: the inner part of the substantia gelatinosa (lamina IIi), the nucleus proprius and the medial parts of laminae V and VI. We also observed considerable labeling in lamina VII and in ventral horn motoneurons. We did not record an increased number of Fos-like immunoreactive neurons in lamina I, in the outer substantia gelatinosa (lamina IIo), or in the lateral, reticulated portion of lamina V, regions that contain neurons predominantly responsive to noxious stimulation. Unilateral sensory deafferentation of the forelimb, by multiple dorsal rhizotomies, significantly decreased the number of Fos-like immunoreactive neurons in the ipsilateral spinal cord, suggesting that afferent input contributed to the walking-induced pattern of labeling. In rats that walked on the Rota-Rod, we also recorded increased labeling in the dorsal column nuclei. Unilateral cervical deafferentation reduced the labeling in the cuneate nucleus; this reduction was paralleled by decreased cytochrome oxidase activity. Finally, we found that there was a significant increase in the number of Fos-like immunoreactive neurons in the cerebellum of rats that walked on the Rota-Rod. Northern blot analysis revealed that the increase in Fos-like immunoreactivity was associated with an increase in c-fos messenger RNA. The pattern of labeling observed in the rats that walked on the Rota-Rod was distinct from that observed when rats are exposed to a noxious stimulus [Presley et al. (1990) J. Neurosci. 10, 323-335]. This result reinforces the conclusion that by monitoring the evoked expression of the c-fos proto-oncogene, it is possible to identify unique populations of neurons that are specifically related to the modality of the stimulus or to behaviour occurring during the stimulus presentation.