In rats with persistent pain, spinal group I metabotropic glutamate receptor (mGluR) activity has been shown to be pronociceptive, whereas spinal group II/III activity is anti-nociceptive. In brain, group I mGluR activity produces positive feedback effects on glutamate release, whereas group II/III activity produces negative feedback effects. It is unknown whether the nociceptive versus anti-nociceptive effects of spinal group I versus group II/III mGluR activity depend on differential regulation of spinal glutamate release. Here, we used behavioral nociceptive testing and in vivo microdialysis to assess the effect of intrathecal treatment with group I mGluR antagonists [cyclopropan[b] chromen-1a-carboxylate, (CPCCOEt), 2-methyl-6-(phenylethynyl) pyridine (MPEP)] or groups II [aminopyrrolidine-2R,4R-dicarboxylate (APDC)] and III [l-2-amino-4-phosphonobutyrate (l-AP4)] mGluR agonists or vehicle, on nociception and noxious stimulus-induced increases in glutamate release in the spinal cord dorsal horn of rats with a chronic constriction injury (CCI) of the sciatic nerve or hind paw injection of complete Freund's adjuvant (CFA). None of the treatments significantly influenced basal spinal glutamate concentrations in either CCI or CFA rats. In CCI rats, formalin-induced nociception and increases in spinal glutamate concentrations were significantly attenuated by pre-treatment with CPCCOEt, MPEP, APDC, or l-AP4. In CFA rats, capsaicin-induced increases in nociception and spinal glutamate concentrations were significantly attenuated by pre-treatment with CPCCOEt, MPEP, or APDC, but not l-AP4. This study demonstrates that group I antagonists and group II/III mGluR agonists attenuated the enhanced nociception and noxious stimulus-induced glutamate release in spinal cord dorsal horn of CCI and/or CFA rats in vivo, and suggests a possible mechanism for their anti-hyperalgesic effects.