To determine the morphological relationships among extensor digitorum longus (EDL), tibialis anterior (TA), and semitendinosus (St) motor nuclei in the spinal cord of the cat, these nuclei were retrogradely labeled with three different fluorescent tracers. The fluorochromes--bisbenzimide, nuclear yellow, and propidium iodide--were applied by intramuscular injection or soaking the muscle nerve. The positions of the labeled motor nuclei were bilaterally symmetrical. The EDL and TA motoneurons were located in close proximity to one another, in the lateral regions of lamina IX in spinal segments L6 and L7. Although the boundaries of each nucleus were tightly opposed, the EDL and TA motor nuclei overlapped minimally, with the somata of EDL motoneurons positioned dorsal to those of TA. The St motor nucleus was located ventromedial to that of EDL and extended from the caudal portion of L6 through S1. Supplemental studies of the reflex effects evoked in EDL, TA, and St muscles by cutaneous nerve stimulation provided physiological observations that may be related to these anatomical results.