Polymorphonuclear leukocytes (PMN) can release opioid peptides which bind to opioid receptors on sensory neurons and inhibit inflammatory pain. This release can be triggered by chemokine receptor 1/2 (CXCR1/2) ligands. Our aim was to identify the granule subpopulation containing opioid peptides and to assess whether MAPK mediate the CXCR1/2 ligand-induced release of these peptides. Using double immunofluorescence confocal microscopy, we showed that beta-endorphin (END) and Met-enkephalin (ENK) were colocalized with the primary (azurophil) granule markers CD63 and myeloperoxidase (MPO) within PMN. END and ENK release triggered by a CXCR1/2 ligand in vitro was dependent on the presence of cytochalasin B (CyB) and on p38 MAPK, but not on p42/44 MAPK. In addition, translocation of END and ENK containing primary granules to submembranous regions of the cell was abolished by the p38 MAPK inhibitor SB203580. In vivo CXCL2/3 reduced pain in rats with complete Freund's adjuvant (CFA)-induced hindpaw inflammation. This effect was attenuated by intraplantar (i.pl.) antibodies against END and ENK and by i.pl. p38 MAPK inhibitor treatment. Taken together, these findings indicate that END and ENK are contained in primary granules of PMN, and that CXCR1/2 ligands induce p38-dependent translocation and release of these opioid peptides to inhibit inflammatory pain.