Identification of chemokine receptors and their associated ligands is crucial to the understanding of most immune reactions. Three human chemokines [I‐309, thymus and activation‐regulated chemokine (TARC) and macrophage inflammatory protein‐1β (MIP‐1β)] have been reported to be ligands for CC‐chemokine receptor 8 (CCR8). In this report, we present evidence that TARC and MIP‐1β did not bind to or induce chemotaxis through CCR8 on a stable transfected cell line (1D‐21) and did not bind to CCR8 on in vitro differentiated human CD4⁺ Th₂ cell cultures. Also, I‐309‐dependent calcium mobilization in 1D‐21 cells and in Th₂ cells was desensitized by I‐309 but not by MIP‐1β or TARC. These results provide strong evidence that, at physiologically relevant concentrations, I‐309 is the only known human ligand for CCR8. These data also provide a framework for suggesting minimum requirements for the assignment of chemokine receptor‐ligand pairs.