The persistent sodium current (I(Na(P))) has been implicated in the regulation of synaptic integration, intrinsic membrane properties, and rhythm generation in many types of neurons. We characterized I(Na(P)) in commissural interneurons (CINs) in the neonatal (postnatal days 0-3) mouse spinal cord; it is activated at subthreshold potentials, inactivates slowly, and can be blocked by low concentrations of riluzole. The role of I(Na(P)) in locomotor pattern generation was examined by applying riluzole during fictive locomotion induced by NMDA, serotonin, and dopamine or by stimulation of the cauda equina. Blockade of I(Na(P)) has marginal effects on the locomotion frequency but progressively weakens the rhythmic firing and locomotor-related membrane oscillation of CINs and motoneurons (MNs) and the locomotor-like bursts in ventral roots, until the motor pattern ceases. Riluzole directly affects the intrinsic firing properties of CINs and MNs, reducing their ability to fire repetitively during tonic depolarizations and raising their spike threshold. At the same time, riluzole has little effects on the strength of spike-evoked synaptic transmission onto CINs and MNs. Our results suggest that I(Na(P)) is essential for the generation of the locomotor pattern and acts in part by regulating the frequency of interneuron firing in the central pattern generator for locomotion.