The effects of selected brainstem lesions on controlled treadmill locomotion produced by stimulation of the mesencephalic locomotor region (MLR) in postmamillary cats were determined in these experiments. The importance for the initiation of locomotion of projections from the MLR to rostral brainstem structures, described in a preceding paper, were examined by selective lesioning or by adjusting the level of the decerebration. The role played by the lateral vestibulospinal tract (LVST) in the initiation of locomotion was examined by lesioning Deiters' nucleus bilaterally. Contrary to previous claims, the results of the present experiments show that areas of the brainstem rostral to the MLR are not required for the initiation of locomotion by MLR stimulation. This finding eliminates the ventral tegmental area of Tsai and the substantia nigra, both implicated in the initiation of locomotion, as required participants in MLR stimulated locomotion. Bilateral Deiters' nucleus (DN) lesions did not significantly affect the initiation of locomotion from the MLR, nor did such lesions alter in a systematic fashion the amplitude or timing of EMG activity in flexor or extensor muscles of the hindlimb during MLR evoked walking. Joint angle changes during the locomotor cycle were also essentially unaltered by DN lesions. The significance of these findings regarding the brainstem structures which must be involved in the initiation of locomotion are discussed.