The circuits that generate rhythmic locomotor activities are located in the ventromedial area of the lumbar spinal cord and comprise commissural interneurons necessary for left-right alternation during walking movements. In this study we injected biotinylated dextran amine (BDA) into the ventromedial gray matter of the lumbar spinal cord of neonatal rats to label commissural interneurons. Anterogradely labeled axons arose from the site of injection, crossed the midline in the anterior commissure and arborized extensively in the contralateral ventral horn of the spinal cord. The presence of neurotransmitter systems in labeled axon terminals of commissural interneurons was investigated by using antibodies raised against specific transmitter-related proteins. Boutons potentially containing inhibitory amino acids were identified by applying glutamic acid decarboxylase (GAD65/67) and glycine transporter 2 antibodies. Out of 1146 BDA-labeled axon terminals, 663 boutons were assumed on this basis to be inhibitory; 76% of these terminals were immunoreactive for glycine transporter, 53% were immunoreactive for GAD and about 30% of inhibitory boutons might contain both inhibitory amino acids. Boutons potentially containing putative excitatory neurotransmitter were revealed with antibodies raised against vesicular glutamate transporters 1 and 2. Out of 590 BDA-labeled boutons about one fourth (158) were immunoreactive for glutamate transporters. These mammalian commissural interneurons are compared to the glycinergic commissural interneurons in the swimming CPGs of lamprey and the Xenopus tadpole. Our results show that commissural interneurons in the mammalian spinal cord form a heterogeneous group including glutamatergic excitatory and GABAergic and glycinergic inhibitory neurons.