The CXCR4 chemokine receptor is a Gi protein-coupled receptor that triggers multiple intracellular signals in response to SDF-1. In this study, we explored the role played by the third intracellular loop (ICL3) of CXCR4 in SDF-1-mediated signaling using human embryonic kidney (HEK)293 cells stably expressing wild type or a mutated form of CXCR4. We demonstrated here that ICL3 is specifically involved in Gi-dependent signals.