Using an in vitro preparation from neonatal rat spinal cord, we compared the motor patterns induced by three putative locomotion-inducing substances. N-Methyl-D,L-aspartate (NMA) induced rhythmic hindlimb nerve activity in 17/20 preparations that was characterized by: (a) side-to-side alternation, but co-activated intralimb flexor-extensor pairs in 29%; (b) bilateral co-activation of all flexors and extensors in 24%; and c) rhythmic but poorly coordinated activity in 35%. Acetylcholine induced rhythmic activity in 34/35 preparations, which in 68% of animals was characterized by side-to-side alternation of co-activated intralimb flexor-extensor pairs. Only rarely did NMA (2/20 trials) and acetylcholine (1/35 trials) induce sustained ENG patterns compatible with hindlimb stepping. Serotonin, however, induced rhythmic activity in 22/24 preparations that was consistent with locomotion in intact rats in 13/22 (59%). These findings demonstrate that exogenously applied neurochemicals induce a variety of in vitro motor rhythms although some substances preferentially activate specific patterns. The results also highlight the importance of monitoring flexor and extensor activity from both hindlimbs in order to distinguish locomotor-like patterns from other types of neurochemically-induced rhythms.