Ligand binding to a chemokine receptor triggers signaling events through heterotrimeric G-proteins. The mechanisms underlying receptor-mediated G-protein activation in the heterogeneous microenvironments of the plasma membrane are unclear. Here, using live-cell fluorescence resonance energy transfer imaging to detect the proximity between CXCR1-cyan fluorescent protein (CFP) and fluorescence probes that label lipid raft or non-lipid raft microdomains and using fluorescence recovery after photobleaching analysis to measure the lateral diffusion of CXCR1-CFP, we found that interleukin-8 induces association between the receptors and lipid raft microenvironments. Disruption of lipid rafts impaired G-protein-dependent signaling, such as Ca 2؉ responses and phosphatidylinositol 3-kinase activation, but had no effect on ligand-binding function and did not completely abolish ligand-induced receptor phosphorylation. Our results suggest a novel mechanism by which ligand binding to CXCR1 promotes lipid raft partitioning of receptors and facilitates activation of heterotrimeric G-proteins.Chemokine receptors belong to a family of seven-transmembrane G-protein-coupled receptors (GPCRs) that are differentially expressed by a number of immune and nonimmune cell populations and mediate cell responses to a family of soluble chemoattractant molecules called chemokines (22,29,45). Chemokines and their receptors control the trafficking of leukocytes and T and B lymphocytes. Cell movement and positioning, two fundamental properties of immune cells, play critical roles in many processes, including inflammation, allergy, and T-and B-cell and dendritic cell interactions that are necessary for self-tolerance and immune responses to various pathogens (10,21,27,42). Extracellular chemokines binding to cell surface receptors initiate dissociation of heterotrimeric G-proteins into G␣i and G␥ subunits. These subunits, in turn, induce downstream intracellular signaling components to generate biochemical responses (29,42). CXCR1 is one of the human chemokine receptors that is selectively expressed in neutrophils and directs leukocytes to sites of inflammation (2, 47). Several chemokines, such as interleukin-8 (IL-8), and acP-2, bind to CXCR1 and activate G-protein-dependent intracellular signaling pathways, for example, Ca 2ϩ influx and phosphatidylinositol 3-kinase (PI3K) activation, and also trigger G-protein-independent CXCR1 phosphorylation (2, 47).The plasma membranes of eukaryotic cells consist of a complex assembly of various lipids and proteins that are distributed in regions of distinct lipid microenvironments, known as lipid raft or non-lipid raft microdomains (8,15,24,32,40,43). Lipids in rafts possess long and saturated fatty acyl chains and are organized in a tightly packed, liquid-ordered manner, whereas non-lipid raft microdomains contain shorter, unsaturated fatty acyl chains and are in a loosely packed, disordered manner (8,15,24,32,40,43). Lipid rafts are defined as microdomains that are enriched in cholesterol, glycosphingo...