The chemokine IP-10 (interferon-inducible protein of 10 kDa, CXCL10) binds the G protein-coupled receptor CXCR3, which is found mainly on activated T cells and NK cells, and plays an important role in Th1-type inflammatory diseases. IP-10 also binds to glycosaminoglycans (GAGs), an interaction thought to be important for its sequestration on endothelial and other cells. In this study, we performed an extensive mutational analysis to identify the CXCR3 and heparin binding sites of murine IP-10. The mutants were characterized for heparin binding, CXCR3 binding, and the ability to induce chemotaxis, Ca(2+) flux, and CXCR3 internalization. Double mutations neutralizing adjacent basic residues at the C terminus did not lead to a significant reduction in heparin binding, indicating that the main heparin binding site of IP-10 is not along the C-terminal alpha helix. Alanine exchange of Arg-22 had the largest effect on heparin binding, with residues Arg-20, Ile-24, Lys-26, Lys-46, and Lys-47 further contributing to heparin binding. A charge change mutation of Arg-22 resulted in further reduction in heparin binding. The N-terminal residue Arg-8, preceding the first cysteine, was critical for CXCR3 signaling. Mutations of charged and uncharged residues in the loop regions of residues 20-24 and 46-47, which caused reduced heparin binding, also resulted in reduced CXCR3 binding and signaling. CXCR3 expressing GAG-deficient Chinese hamster ovary cells revealed that GAG binding was not required for IP-10 binding and signaling through CXCR3, which suggests that the CXCR3 and heparin binding sites of IP-10 are partially overlapping.