Intrathecal (i.t.) administration of the group I metabotropic glutamate receptor (mGluR) agonist (RS)-3,5-dihydroxyphenylglycine ((RS)-3,5-DHPG) to rats produces an immediate display of spontaneous nociceptive behaviors (SNBs) persisting for up to 10 h after injection (NeuroReport 7 (1996) 2743). The mechanisms underlying these behavioral effects are not entirely understood but may include enhanced release of glutamate within the dorsal horn of the spinal cord. The current experiments used microdialysis in awake moving animals to test: (1), whether i.t. (S)-3,5-DHPG increases the local release of glutamate at doses that also induce SNBs; and (2), whether the effects on glutamate release (as well as SNBs) can be blocked by pretreatment with the mGluR5 selective antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP). Male Sprague-Dawley rats were implanted with a microdialysis probe inserted into the i.t. space of the spinal cord (J. Neurosci. Methods 62 (1995) 43) and then tested under i.t. drug conditions (0.01, 0.1 and 1 mM (S)-3,5-DHPG) following a 2-3 day recovery period. As predicted, local application of (S)-3,5-DHPG via the microdialysis probe increased the release of glutamate in a dose-dependent manner. Significant SNBs were also noted in the 0.1 and 1 mM groups in a manner paralleling the onset and duration of the glutamate response. Pretreatment with MPEP (55 mg/kg, intraperitoneally) blocked glutamate release to the 0.1 mM dose of (S)-3,5-DHPG, and also decreased the proportion of animals displaying SNBs in this dose group. No effects of MPEP were seen against the higher dose of (S)-3,5-DHPG (1 mM). These results suggest that stimulation of spinal mGluR5 leads to glutamate release within the spinal cord, a response that may in part account for the nociceptive behaviors evoked by i.t. (S)-3,5-DHPG.