Cross-desensitization between micro-opioid receptor agonists and CC chemokines was shown to occur in immune cells and in the central nervous system. However, these cells do not permit examination of potential mechanisms at cellular levels due to low levels and mixed populations of receptors. In this study, we investigated possible interactions and biochemical mechanisms of cross-desensitization between the mu-opioid and chemokine CCR5 receptors coexpressed in Chinese hamster ovary (CHO) cells. Hemagglutinin (HA)-tagged micro-opioid receptor coimmunoprecipitated with FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys)-tagged chemokine receptor CCR5 in cells expressing the two receptors, but not in a mixture of cells transfected with one of the two receptors, indicating that the two receptors form heterodimers. Treatment with the mu-opioid receptor agonist DAMGO ([D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin), the chemokine RANTES (Regulated on Activation, Normal T cell-Expressed and -Secreted) (CCL5), or both, did not affect the level of coimmunoprecipitation. DAMGO and RANTES (CCL5) induced chemotaxis in CHO cells coexpressing both receptors, and preincubation with either DAMGO or RANTES (CCL5) profoundly inhibited chemotaxis caused by the other. DAMGO pretreatment enhanced phosphorylation of the chemokine CCR5 receptor and reduced RANTES (CCL5)-promoted [35S]GTP gamma S binding. Conversely, RANTES (CCL5) preincubation slightly increased phosphorylation of the mu-opioid receptor and significantly reduced DAMGO-induced [35S]GTP gamma S binding. These results indicate that activation of either receptor affected G protein coupling of the other, likely due to enhanced phosphorylation of the receptor. Heterodimerization between the two receptors may contribute to the observed cross-desensitization.