Leukocytes counteract inflammatory pain by releasing opioid peptides, which bind to opioid receptors on peripheral sensory neurons. In the early phase of inflammation, polymorphonuclear cells (PMN) are the major source of opioids. Their recruitment is governed by ligands at the chemokine receptor CXCR2. Here, we examined whether chemokines can also induce opioid peptide secretion from PMN and thus inhibit inflammatory pain. In rats with hindpaw inflammation, intraplantar injection of CXCL2/3, but not of the CXCR4 ligand CXCL12, elicited naloxone-reversible (i.e., opioid receptor mediated) mechanical and thermal analgesia, which was abolished by systemic PMN depletion. Both CXCR1/2- and CXCR4-ligands induced PMN chemotaxis, but only CXCR1/2 ligands triggered opioid release from human and rat PMN in vitro. This release was unaltered by extracellular Ca²⁺ chelation, was mimicked by thapsigargin and was blocked by inhibitors of the inositol 1,4,5-triphosphate receptor (IP₃) and by intracellular Ca²⁺ chelation, indicating that it required Ca²⁺ from intracellular but not extracellular sources. Furthermore, release was partially reduced by phosphoinositol-3-kinase (PI3K) inhibitors. Adoptive transfer of allogenic PMN into PMN-depleted rats reconstituted CXCL2/3-induced analgesia, which was inhibited by prior ex vivo chelation of intracellular Ca²⁺. These findings demonstrate that, beyond cell recruitment, CXCR2 ligands induce Ca²⁺-regulated opioid release from PMN and thereby inhibit inflammatory pain in vivo.--Rittner, H. L., Labuz, D., Schaefer, M., Mousa, S. A., Schulz, S., Schäfer, M., Stein, C., and Brack, A. Pain control by CXCR2 ligands through Ca²⁺-regulated release of opioid peptides from polymorphonuclear cells.