We examined the effect of p38 mitogen-activated protein kinase (MAPK) inhibitors in models of nociception and correlated this effect with localization and expression levels of p38 MAPK in spinal cord. There was a rapid increase in phosphorylated p38 MAPK in spinal cord following intrathecal administration of substance P or intradermal injection of formalin. Immunocytochemistry revealed that phosphorylated p38 MAPK-immunoreactive cells were predominantly present in laminae I-IV of the dorsal horn. Double-staining with markers for neurons, microglia, astrocytes and oligodendrocytes unexpectedly revealed co-localization with microglia but not with neurons or other glia. Pretreatment with p38 MAPK inhibitors (SB20358 or SD-282) had no effect on acute thermal thresholds. However, they attenuated hyperalgesia in several nociceptive models associated with spinal sensitization including direct spinal activation (intrathecal substance P) and peripheral tissue inflammation (intraplantar formalin or carrageenan). Spinal sensitization, manifested by enhanced expression of cyclo-oxygenase-2 and inflammation-induced appearance of Fos-positive neurons, was blocked by pretreatment, but not post-treatment, with p38 MAPK inhibitors. Taken together, these results indicate that spinal p38 MAPK is involved in inflammation-induced pain and that activated spinal microglia play a direct role in spinal nociceptive processing.