Fast glutamatergic transmission via ionotropic receptors is critical for the generation of locomotion by spinal motor networks. In addition, glutamate can act via metabotropic glutamate receptors (mGluRs) to modulate the timing of ongoing locomotor activity. In the present study, we investigated whether mGluRs also modulate the intensity of motor output generated by spinal motor networks. Application of the group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) reduced the amplitude and increased the frequency of locomotor-related motoneuron output recorded from the lumbar ventral roots of isolated mouse spinal cord preparations. Whole cell patch-clamp recordings of spinal motoneurons revealed multiple mechanisms by which group I mGluRs modulate motoneuron output. Although DHPG depolarized the resting membrane potential and reduced the voltage threshold for action potential generation, the activation of group I mGluRs had a net inhibitory effect on motoneuron output that appeared to reflect the modulation of fast, inactivating Na(+) currents and action potential parameters. In addition, group I mGluR activation decreased the amplitude of locomotor-related excitatory input to motoneurons. Analyses of miniature excitatory postsynaptic currents indicated that mGluRs modulate synaptic drive to motoneurons via both pre- and postsynaptic mechanisms. These data highlight group I mGluRs as a potentially important source of neuromodulation within the spinal cord that, in addition to modulating components of the central pattern generator for locomotion, can modulate the intensity of motoneuron output during motor behavior. Given that group I mGluR activation reduces motoneuron excitability, mGluRs may provide negative feedback control of motoneuron output, particularly during high levels of glutamatergic stimulation.