AbstractThe chemokine receptor CXCR4 and the μ‐opioid receptor (MOR) are G‐protein‐coupled receptors that are essential for normal function of the nervous and immune systems. Several studies have suggested that MOR is a key regulator of CXCR4 in the brain; however, the molecular basis of the opioid–chemokine interaction is not fully understood, and it may involve different mechanisms in neuronal and glial cells. Our previous studies demonstrated that MOR stimulation specifically upregulates the protein ferritin heavy chain – an inhibitor of CXCR4 – in neurons, and suggested that additional mechanisms could be operative in glia. In this study, we investigated CXCR4 function in brains and astroglial cultures deprived of MOR. Reduced coupling of CXCR4 to G‐proteins was found in brain slices and tissue homogenates of MOR−/− mice as compared with wild‐type controls. CXCR4‐induced signaling was also reduced in glial cultures from MOR−/− mice, as shown by analysis of CXCR4 downstream targets (Akt and ERK1/2). Pharmacological studies with δ‐opioid receptor (DOR)‐specific ligands suggested that DOR–CXCR4 interactions are implicated in the inhibition of CXCR4 in MOR‐deficient cells both in vitro and in vivo. Moreover, increased CXCR4/DOR co‐immunoprecipitation was found in brain tissue and cultured glia from MOR−/− mice. Importantly, CXCR4 function was restored by pretreatment with a DOR antagonist. Overall, these findings indicate that DOR plays a crucial role in the regulation of CXCR4 in glia, probably via silent receptor heterodimers. The data also suggest that the opiate system interferes with normal CXCR4 function in different ways, depending on receptor subtypes.