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 gray 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 with motoneurons, interneurons were more excitable, because of higher input resistance and membrane time constant, and displayed lower firing frequency arising from 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 Ca(2+) currents. We further used in vitro isolated spinal cord preparations to investigate the contribution of I(NaP) to locomotor pattern. Application of riluzole (10 muM) 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 arise from a specific blockade of I(NaP) because 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 I(NaP)-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.