Injections of a tritiated amino acid-fluorescent dye mixture were made unilaterally into the area of the mesencephalic locomotor region (MLR). After allowing for retrograde and anterograde transport, the same site was electrically stimulated to induce locomotion on a treadmill following a precollicular-postmamillary transection. The tritiated amino acid transported anterogradely primarily was found autoradiographically to descend in the area of Probst's tract and to ascend to the centremedian nucleus (CM) of the thalamus. Neurons labeled retrogradely by the fluorescent dye in the same injection-stimulation site were observed in the substantia nigra, entopeduncular nucleus, sub- and hypothalamus and amygdala. In subsequent experiments, injections of fluorescent tracers were made into the area of Probst's tract and CM. Neurons in the mesencephalic trigeminal root, cuneiform nucleus, nucleus tegmenti pedunculopontinus (NTPP), dorsal locus coeruleus and lateral central gray were labeled from Probst's tract injections. Neurons in medial and lateral central gray, as well as NTPP, were labeled from CM injections.