5-HT₇ receptors have been implicated in the control of locomotion. Here we use 5-HT₇ receptor knockout mice to rigorously test whether 5-HT acts at the 5-HT₇ receptor to control: 1) locomotor-like activity in the neonatal mouse spinal cord in vitro and 2) voluntary locomotion in adult mice. We found that 5-HT applied onto in vitro spinal cords of 5-HT₇+/+ mice produced locomotor-like activity that was disrupted and subsequently blocked by the 5-HT₇ receptor antagonist SB-269970. In 5HT₇-/- mice, 5-HT produced either uncoordinated rhythmic activity or resulted in synchronous discharges of the ventral roots. SB-269970 had no effect on 5-HT induced rhythmic activity in the 5HT₇-/- mice. In adult in vivo experiments, SB-269970 applied directly to the spinal cord consistently disrupted locomotion and produced hyper-extension of the hindlimbs in 5-HT₇+/+ but not 5-HT₇-/- mice. Disrupted EMG activity produced by SB-269970 in vivo was similar to the uncoordinated rhythmic activity produced by the drug in vitro. Moreover, 5-HT₇-/- mice displayed greater maximal extension at the hip and ankle joints than 5-HT₇+/+ animals during voluntary locomotion. These results suggest that spinal 5-HT₇ receptors are required for the production and coordination of 5-HT-induced locomotor-like activity in the neonatal mouse and are important for the coordination of voluntary locomotion in adult mice. We conclude that spinal 5-HT₇ receptors are critical for alternating activity during locomotion.