Electrophysiological and morphological properties of genetically identified spinal interneurons were examined to elucidate their possible contribution to locomotor-like rhythmic activity in 1- to 4-day-old mice. In the transgenic mice used in our study, the HB9 promotor controlled the expression of the reporter gene enhanced green fluorescent protein (eGFP), giving rise to GFP+ motoneurons and ventral interneurons. However, only motoneurons and a small group of bipolar, GFP+ interneurons expressed the HB9 protein. The HB9(+)/GFP+ interneurons were clustered close to the medial surface in lamina VIII along segments L1-L3. The correlation between activity pattern in these visually identified interneurons and motoneuron output was examined using simultaneous whole cell and ventral root recordings. Neurochemically induced rhythmic membrane depolarizations in HB9/GFP interneurons were synchronous with ventral root rhythms, indicating that the interneurons received synaptic inputs from rhythm-generating networks. The frequency of excitatory postsynaptic currents significantly increased during ventral root bursts, but there was no change in the frequency of inhibitory postsynaptic currents during the cycle period. These data implied that HB9/GFP interneurons received primarily excitatory inputs from rhythmogenic interneurons. Neurobiotin-filled axon terminals were in close apposition to other neurons in the cluster and to motoneuron dendrites, raising the possibility that HB9/GFP interneurons formed synaptic connections with each other and with motoneurons. The expression of the vesicular glutamate transporter 2 in axon terminals of HB9/GFP interneurons indicated that these were glutamatergic interneurons. Our findings suggest that the visually identified HB9/GFP interneurons are premotor excitatory interneurons and putative constituents of networks generating locomotor rhythms in the mammalian spinal cord.