CCR1 and CCR3 are seven-transmembrane domain G protein-coupled receptors specific for members of the CC chemokine subgroup of leukocyte chemoattractants. Both have been implicated in the inflammatory response, and CCR3, through its expression on eosinophils, basophils, and Th2 lymphocytes, may be especially important in allergic inflammation. CCR1 and CCR3 are 54% identical in amino acid sequence and share some ligands but not others. In particular, macrophage inflammatory protein 1alpha (MIP-1alpha) is a ligand for CCR1 but not CCR3, and eotaxin is a ligand for CCR3 but not CCR1. To map ligand selectivity determinants and to guide rational antagonist design, we analyzed CCR1:CCR3 chimeric receptors. When expressed in mouse pre-B cells, chimeras in which the N-terminal extracellular segments were switched were both able to bind both MIP-1alpha and eotaxin, but in each case, binding occurred via separate sites. Nevertheless, neither MIP-1alpha nor eotaxin were effective agonists at either chimeric receptor in either calcium flux or chemotaxis assays. These data are consistent with a multi-site model for chemokine-chemokine receptor interaction in which one or more subsites determine chemokine selectivity, but others are needed for receptor activation. Agents that bind to the N-terminal segments of CCR1 and CCR3 may be useful in blocking receptor function.