Commissural projections are required for left-right coordination during locomotion. However, their role, if any, in rhythm production is unknown. The present study employs the neonatal rat in vitro brainstem-spinal cord model to examine the rostrocaudal distribution of locomotor-related commissural projections and investigate whether commissural connections are required for the generation of hindlimb rhythmic activity in response to electrical stimulation of the brainstem. Midsagittal lesions were made at a wide range of rostrocaudal levels. Locomotor-like activity persisted in some preparations despite midsagittal lesions extending from C1 to the mid-L1 level, or from the conus medullaris to the T12/13 junction. In some preparations midsagittal lesions throughout the entire spinal cord had no effect on locomotor-like activity, provided two or three contiguous segments remained intact. Those bridging segments had to include the T13 and/or L1 levels. These observations suggested that commissural projections in the thoracolumbar junction region were critical. However, locomotor-like activity was also elicited in preparations with limited midsagittal lesions focused on the thoracolumbar junction (T12 through L1 or L2 inclusive). In other experiments, locomotor-like activity was evoked by bath-applied 5-HT and NMDA. Appropriate side-to-side coordination was observed, even when only one segment remained bilaterally intact. Commissural projections traversing the thoracolumbar junction region were most effective. In combination, these results suggest that locomotor-related commissural projections are redundantly distributed along a bi-directional gradient that centers on the thoracolumbar junction. This commissural system not only provides a robust left-right coordinating mechanism, but also supports locomotor rhythm generation in response to brainstem stimulation.