Opioid and chemokine receptor distribution has been described to overlap in white blood cells such as neutrophils and monocytes. Increasing evidence also suggests that opioid ligands can alter immune response by modulating chemotaxis and that chemokine receptors play a role in neurone migration. In this study we have shown that the DOR and CXCR2 receptors can form heterodimers using techniques including co-immunoprecipitation, FRET and BRET. We then applied the Dimerscreen^TM technology to investigate DOR/CXCR2 heterodimer pharmacology. This system is based on a complementary pair of mutated fusion proteins, MycCXCR2^I148EG_i2^C351I and FlagDORG_i2^G204AC351I that are unable to signal unless the receptors form a dimer. This approach allowed us to investigate selectively CXCR2/DOR heterodimer response to ligands. We observed that the CXCR2 antagonist SB225002 enhanced the function of agonists at DOR when this receptor was co-expressed with CXCR2 but had no effect on DOR when this receptor was expressed separately. This ‘off target’ allosteric effect was observed for both peptidic and non-peptidic opioid agonists.