We describe here the robust synchronization of motor neurons at a millisecond time scale during locomotor activity in the neonatal rat. Action potential activity of motor neuron pairs was recorded extracellularly using tetrodes during locomotor activity in the in vitro neonatal rat spinal cord. Approximately 40% of motor neuron pairs recorded in the same spinal segment showed significant synchronization, with the duration of the central peak in cross-correlograms between motor neurons typically ranging between ∼ 30 and 100 msec. The percentage of synchronized motor neuron pairs was considerably higher for pairs with similar locomotor-related activity and strong rhythmic modulation. We also found synchronization between the activities of different motor pools, even if located several segments apart. Such distant synchronization was abolished in the absence of chemical synapses, although local coupling between motor neurons persisted. On the other hand, both local and distant coupling between motor neurons were preserved after antagonism of gap junction coupling between motor neurons. These results demonstrate that motor neuron activity is strongly synchronized at a millisecond time scale during the production of locomotor activity in the neonatal rat. These results also demonstrate that chemical synaptic inputs, in addition to electrical synapses, contribute to this synchronization, suggesting the existence of multiple mechanisms underlying motor neuron synchronization in the neonatal rat. The fast synchronization described here might be involved in activity-dependent processes during development or in the coordination of individual motor neurons into a functional population underlying behavior.