Commissural inhibitory interneurons (INs) are integral components of the locomotor circuitry that coordinate left-right motor activity during movements. Wehave shown that GABA-mediated synaptic transmission plays a key role in generating alternating locomotor-like activity in the mouse spinal cord (Hinckley et al., 2005a). The primary objective of our study was to determine whether properties of lamina VIII (LVIII) GAB Aergic INs in the spinal cord of GAD67::GFP transgenic mice fit the classification of rhythm-coordinating neurons in the locomotor circuitry. The relatively large green fluorescent protein-expressing (GFP+) INs had comparable morphological and electrophysiological properties, suggesting that they comprised a homogenous neuronal population. They displayed multipolar and complex dendritic arbors in ipsilateral LVII-LVIII, and their axonal projections crossed the ventral commissure and branched into contralateral ventral, medial, and dorsal laminae. Putative synaptic contacts evident as bouton-like varicosities were detected in close apposition to lateral motoneurons, Renshaw cells, other GFP+ INs, and unidentified neurons. Exposure to a rhythmogenic mixture triggered locomotor-like rhythmic firing in the majority of LVIII GFP+ INs. Their induced oscillatory activity was out-of-phase with bursts of contralateral motoneurons and in-phase with bouts of ipsilateral motor activity. Membrane voltage oscillations were elicited by rhythmic increases in excitatory synaptic drive and might have been augmented by three types of voltage-activated cationic currents known to increase neuronal excitability. Based on their axonal projections and activity pattern, we propose that this population of GABAergic INs forms a class of local commissural inhibitory interneurons that are integral component of the locomotor circuitry.