Spinal circuits known as central pattern generators maintain vertebrate locomotion. In the lamprey, the contralaterally alternating ventral root activity that defines this behavior is driven by ipsilateral glutamatergic excitation (Buchanan and Grillner, 1987) coupled with crossed glycinergic inhibition (Buchanan, 1982; Alford and Williams, 1989). These mechanisms are distributed throughout the spinal cord. Glutamatergic excitatory synapses activate AMPA and NMDA receptors known to be necessary for the maintenance of the locomotor rhythm. Less is known of the role and location of metabotropic glutamate receptors (mGluRs), although group I mGluRs enhance transmitter release at giant synapses in the lamprey spinal cord, whereas group II/III receptors may inhibit release. In this study we show that group I mGluR antagonists block fictive locomotion, a neural correlate of locomotion, by acting at the presynaptic terminal. Under physiological conditions, synaptically released glutamate activates presynaptic group I mGluRs (autoreceptors) during the repetitive activation of glutamatergic terminals. The resulting rise in [Ca2+]i caused by the release from presynaptic intracellular stores is coincident with an enhancement of synaptic transmission. Thus, blocking mGluRs reduces glutamate release during the repetitive activity that is characteristic of locomotion, leading to the arrest of locomotor activity. We found the effects of group I mGluRs on locomotion to be inconsistent with a postsynaptic effect on the central pattern generator. Consequently, the activation of metabotropic glutamate autoreceptors is necessary to maintain rhythmic motor output. Our results demonstrate the role of presynaptic mGluRs in the physiological control of movement for the first time.