This study examines whether propriospinal transmission contributes to descending propagation of the brainstem locomotor command signal in the in vitro neonatal rat spinal cord. Using double bath partitions, synaptic transmission was suppressed in the cervicothoracic region while monitoring locomotor-like activity on lumbar ventral roots evoked by either chemical or electrical stimulation of the brainstem. Locomotor-like activity induced by electrical stimulation was more stable (cycle period coefficient of variation (CV) 11.7 ± 6.1%) than the rhythm induced by chemical stimulation (CV 31.3 ± 6.4%). Ca2+-free bath solution, elevated Mg2+ ion concentration, excitatory amino acid receptor antagonists (AP5 and/or CNQX), and the muscarinic receptor antagonist, atropine, were used in attempts to block synaptic transmission. Each of these manipulations, except muscarinic receptor blockade, was capable of blocking locomotor-like activity induced by brainstem stimulation. However, locomotor-like activity induced by higher intensity electrical stimulation of the brainstem (1.2–5 times threshold) was relatively refractory to synaptic suppression using AP5 and CNQX, and Ca2+-free solution was more effective if combined with high Mg2+ (15 mM) or EGTA. Enhancement of neuronal excitation in the cervicothoracic region, using Mg2+-free bath solution, facilitated brainstem activation of locomotor-like activity in the lumbar cord, consistent with a propriospinal mechanism of locomotor signal propagation. Blockade of brainstem-induced locomotor-like activity was related to the number of cervicothoracic segments exposed to synaptic suppression, being most effective if five or more segments were included. These results provide direct evidence that propriospinal pathways contribute to bulbospinal activation of the locomotor network in the in vitro neonatal rat brainstem–spinal cord preparation, and suggest that a propriospinal system is recruited in parallel with long direct projections that activate the locomotor network.