The persistent sodium current (I_NaP) is known to play a role in rhythm generation in different systems. Here, we investigated its contribution to locomotor pattern generation in the neonatal rat spinal cord. The locomotor network is mainly located in the ventromedial grey matter of upper lumbar segments. By means of whole-cell recordings in slices, we characterized membrane and I_NaP biophysical properties of interneurons located in this area. Compared to motoneurons, interneurons were more excitable, because of higher input resistance and membrane time constant, and displayed lower firing frequency, because of broader spikes and longer AHPs. Ramp voltage-clamp protocols revealed a riluzole- or TTX-sensitive inward current, presumably I_NaP, three times smaller in interneurons than in motoneurons. However, in contrast to motoneurons, I_NaP mediated a prolonged plateau potential in interneurons after reducing K⁺ and Ca²⁺ currents. We further used in vitro isolated spinal cord preparations to investigate the contribution of I_NaP to locomotor pattern. Application of riluzole (10 µM) to the whole spinal cord or to the upper lumbar segments disturbed fictive locomotion, whereas application of riluzole over the caudal lumbar segments had no effect. The effects of riluzole appeared to be due to a specific blockade of I_NaP, since action potential waveform, dorsal root-evoked potentials and miniature excitatory postsynaptic currents were not affected. This study provides new functional features of ventromedial interneurons with the first description of INaP-mediated plateau potentials, and new insights into the operation of the locomotor network with a critical implication of I_NaP in stabilizing the locomotor pattern.