Lower vertebrates can recover locomotor function after spinal transections, and several classes of neurons in these animals can regenerate. However, no studies have directly tested for functional regeneration of descending command pathways for the initiation of locomotion in the absence of complications from sensory feedback. In the present study, the contribution of regeneration of descending command pathways to the reacquisition of locomotion was examined in behaviorally recovered, spinal-transected larval lampreys using in vitro brain/spinal cord preparations, in which mechanosensory feedback was eliminated. Activation of brainstem command centers produced locomotor activity below a healed transection site and this burst activity was coupled 1:1 with locomotor activity above the transection site. Furthermore, the parameters characterizing locomotor patterns in in vitro preparations were similar to those recorded during locomotion in normal, whole-animals. Additional experiments indicated that descending pathways from the brainstem had regenerated across the healed transection site and could directly activate spinal locomotor networks below the lesion area. These data appear to be the first direct demonstration in a vertebrate of functional regeneration of descending command pathways for the initiation of locomotion.