Locomotion is an essential motor act that for the most part is controlled by neuronal circuits in the spinal cord itself, called central pattern generators (CPGs), although their activity is turned on from centers in the brainstem. Understanding the operation of CPG circuits in mammals has been a significant challenge to neuroscientists over the last 50 years. The CPG for walking generates rhythm, as well as the precise patterns of muscular activity. The neural assembly that is directly involved in generating the locomotor rhythm is completely unknown. There is strong evidence from pharmacological and lesion studies showing that glutamatergic neurons are responsible for this function. Here, I propose to identify these key glutamatergic neuronal CPG circuits. As a first step we will use state-of-the-art RNA-sequencing to obtain the complete transcriptome of glutamatergic subpopulations in the ventral spinal cord of the mouse to define new postnatally expressed molecular markers. To link glutamatergic neuronal subpopulations to the locomotor behavior we will use transgenic mouse systems to incorporate light-activated switches in a cell specific way. These tools will provide a new basis for functional and network studies needed to understand the CPG operation. We also propose to use mouse models with optogenetic switches to provide a molecular identification of the glutamatergic locomotor command systems and their integration in the CPG. Our analysis will provide a unified characterization of the neuronal organization of the mammalian CPG and its immediate control from the brain. Understanding the locomotor CPG and its control in mammals is of outmost importance for improving rehabilitation of spinal cord injured patients and designing new repair strategies.