Inhibitory synaptic transmission is an essential element for the central pattern generator (CPG) for locomotion in the spinal cord. However, the actual role of inhibitory neurons in the mammalian CPG and how they are modulated remains unclear. We examined the synaptic inputs to Renshaw cells (RCs) which provide recurrent inhibition to motoneurons. We performed whole cell recordings from visually identified GABAergic neurons in the lumbar (L2 segment) ventral horn using isolated spinal cord preparations taken from glutamic acid decarboxylase-green fluorescence protein (GAD67-GFP) knock-in mouse neonates (Tamamaki et al. 2003). Animals were humanely killed before isolating the spinal cord. Among the recorded neurons, RCs were identified by 1) electrically stimulating the adjacent ventral root to evoke a short latency EPSP which was blocked by bath-application of a nicotinic receptor blocker mecamylamine or d-tubocurarine with glutamatergic antagonist CNQX (Nishimaru et al. in press), and 2) filling the cell with Alexa-dyes and confirming their expression of calbindin after recording (Carr et al. 1998). Locomotor-like rhythmic activity evoked by bath-application of 5-HT and NMDA was monitored in the L2 ventral root that represents the flexor activity. About 50% of the recorded RCs fired in phase with the ipsilateral L2 rhythm while the other remaining RCs fired out of phase with the ventral root rhtyhm. RCs received both excitatory synaptic inputs and inhibitory synaptic inputs during the locomotor-like rhythmic activity. Blocking the nicotinic receptors by mecamylamine markedly reduced the amplitude of the excitatory synaptic inputs indicating that these inputs are mainly from motoneurons. The inhibitory input, on the other hand, persisted in the presence of the nicotinic blocker suggesting that they are from other sources. Such rhythmic synaptic inputs in RCs could also be observed in a split hemicord indicating that these inputs are from neurons located on the same side. These results suggest that during locomotor activity, RCs are modulated not only by motoneurons but also by the CPG itself.