Previous work established that in vitro bath application of N-methyl-D-aspartic acid (NMDA) promotes locomotor activity in a variety of vertebrate preparations including the neonatal rat spinal cord. In addition, NMDA receptor activation gives rise to active membrane properties that are postulated to contribute to the generation or stabilization of locomotor rhythm. However, earlier studies yield conflicting evidence as to whether NMDA receptors are essential in this role. Therefore, in the present series we examined the effect of NMDA receptor blockade, using D-2-amino-5-phosphono-valeric acid, on locomotor-like activity in the in vitro neonatal rat spinal cord. Locomotor-like activity was induced using 5-hydroxytryptamine (5-HT), acetylcholine, combined 5-HT and NMDA receptor activation, increased K⁺ concentration, or electrical stimulation of the brainstem, and monitored using suction electrode recordings of left and right lumbar ventral root discharge. We also studied the effect on locomotor capacity of selectively suppressing NMDA receptor-mediated active membrane properties; this was achieved by removing Mg²⁺ ions from the bath, which in turn abolishes voltage-sensitive blockade of the NMDA receptor channel. The results demonstrate that although NMDA receptor activation may appear essential for locomotor network operation under some experimental conditions, locomotor-like rhythms can nevertheless be generated in the presence of AP5 if spinal cord circuitry is exposed to appropriate levels of non-NMDA receptor-dependent excitation. Therefore, neither NMDA receptor-mediated non-linear membrane properties nor NMDA receptor activation in general is universally essential for locomotor network activation in the in vitro neonatal rat spinal cord.