The c-fos immunohistochemical method of activity-dependent labeling was used to localize locomotor-activated neurons in the adult cat spinal cord. In decerebrate cats, treadmill locomotion was evoked by electrical stimulation of the mesencephalic locomotor region (MLR). Spontaneous or MLR-evoked fictive locomotion was produced in decerebrate animals paralyzed with a neuromuscular blocking agent. After bouts of locomotion during a 7- to 9-h time period, the animals were perfused and the L3-S1 spinal cord segments removed for immunohistochemistry. Control animals were subjected to the same surgical procedures but no locomotor task. Labeled cells were concentrated in Rexed's laminae III and IV of the dorsal horn and laminae VII, VIII, and X of the intermediate zone/ventral horn after treadmill locomotion. Cells in laminae VII, VIII, and X were labeled after fictive locomotion, but labeling in the dorsal horn was much reduced. In control animals, c-fos labeling was a small fraction of that observed in the locomotor animals. The results suggest that labeled cells in laminae VII, VIII, and X are premotor interneurons involved in the production of locomotion, whereas the laminae III and IV cells are those activated during locomotion due to afferent feedback from the moving limb. c-fos-labeled cells were most numerous in the L5-L7 segments, consistent with the distribution of locomotor activated neurons detected through the use of MLR-evoked field potentials.