Previous studies have shown that a group of ventrally located neurons, designated V2a interneurons, play a key role in maintaining locomotor rhythmicity and in ensuring appropriate left-right alternation during locomotion (Crone et al., 2008, 2009). These V2a interneurons express the transcription factor Chx10. The aim of the present study was to characterize the locomotor-related activity of individual V2a interneurons, their cellular properties, and their detailed anatomical attributes in Chx10-GFP mice. A dorsal horn-removed preparation was developed to allow for visual whole-cell patch recordings from V2a interneurons along the entire lumbar spinal cord while at the same time leaving enough of the spinal cord intact to generate fictive locomotion. During drug-evoked locomotor-like activity, a large proportion of Chx10 cells showed rhythmic firing or membrane potential fluctuations related to either flexor or extensor activity in every lumbar segment. Chx10 cells received predominantly rhythmic excitatory input. Chx10 neurons displayed a wide variety of firing and potential rhythmogenic properties. However, none of these properties was obviously related to the observed rhythmicity during locomotor-like activity. In dual recordings, we found no evidence of Chx10 neuron interconnectivity. Intracellular fills revealed diverse projection patterns with most Chx10 interneurons being local with projections to the central pattern generator and motor neuron regions of the spinal cord and others with long ascending and/or descending branches. These data are compatible with V2a neurons having a role in regulating segmental left-right alternation and ipsilateral motor neuron firing with little effect on rhythm generation.