Commissural interneurons (CINs) are a necessary component of central pattern generators (CPGs) for locomotion because they mediate the coordination of left and right muscle activity. The projection patterns and relative locations of different classes of CINs in the ventromedial part of the rodent lumbar cord have been described (Eide et al. [1999] J Comp Neurol 403:332-345; Stokke et al. [2002] J Comp Neurol 446:349-359; Nissen et al. [2005] J Comp Neurol 483:30-47). However, the distribution and relative prevalence of different CIN neurotransmitter phenotypes in the ventral region of the mammalian spinal cord where the locomotor CPG is localized is unknown. In this study we describe the relative proportions and anatomical locations of putative inhibitory and excitatory CINs in the lumbar spinal cord of newborn mice. To directly visualize potential neurotransmitter phenotypes we combined retrograde labeling of CINs with in situ hybridization against the glycine transporter, GlyT2, or the vesicular glutamate transporter, vGluT2, in wildtype mice and in transgenic mice expressing eGFP driven by the promoters of glutamic acid decarboxylase (GAD) 65, GAD67, or GlyT2. Our study shows that putative glycinergic, GABAergic, and glutamatergic CINs are expressed in almost equal numbers, with a small proportion of CINs coexpressing GlyT2 and GAD67::eGFP, indicating a putative combined glycinergic/GABAergic phenotype. These different CIN phenotypes were intermingled in laminas VII and VIII. Our results suggest that glycinergic, GABAergic, and glutamatergic CINs are the principal CIN phenotypes in the CPG region of the lumbar spinal cord in the newborn mouse. We compare these results to descriptions of CIN neurotransmitter phenotypes in other vertebrate species.