We recently showed that propriospinal neurons contribute to bulbospinal activation of locomotor networks in the in vitro neonatal rat brainstem-spinal cord preparation. In the present study, we examined whether propriospinal neurons alone, in the absence of long direct bulbospinal transmission to the lumbar cord, can successfully mediate brainstem activation of the locomotor network. In the presence of staggered bilateral spinal cord hemisections, the brainstem was stimulated electrically while recording from lumbar ventral roots. The rostral hemisection was located between C1 and T3 and the contralateral caudal hemisection was located between T5 and mid-L1. Locomotor-like activity was evoked in 27% of the preparations, which included experiments with staggered hemisections placed only two segments apart. There was no relation between the likelihood of developing locomotor-like activity and the distance separating the two hemisections or specific level of the hemisections. In some experiments, where brainstem stimulation alone was ineffective, neurochemical excitation of propriospinal neurons (using 5-HT and NMDA) at concentrations subthreshold for producing locomotor-like activity, promoted locomotor-like activity in conjunction with brainstem stimulation. In other experiments, involving neither brainstem stimulation nor cord hemisections, the excitability of propriospinal neurons in the cervical and/or thoracic region was selectively enhanced by bath application of 5-HT and NMDA or elevation of bath K⁺ concentration. These manipulations produced locomotor-like activity in the lumbar region. In total, the results suggest that propriospinal neurons are sufficient for transmission of descending locomotor command signals. This observation has implications for regeneration strategies aimed at restoration of locomotor function after spinal cord injury.