Keratinocyte-derived chemokine (KC or mCXCL1) and macrophage inflammatory protein 2 (MIP2 or mCXCL2) play nonredundant roles in trafficking blood neutrophils to sites of infection and injury. The functional responses of KC and MIP2 are intimately coupled to their interactions with glycosaminoglycans (GAGs). GAG interactions orchestrate chemokine concentration gradients and modulate receptor activity, which together regulate neutrophil trafficking. Here, using NMR, molecular dynamics (MD) simulations, and isothermal titration calorimetry (ITC), we characterized the molecular basis of KC and MIP2 binding to the GAG heparin. Both chemokines reversibly exist as monomers and dimers, and the NMR analysis indicates that the dimer binds heparin with higher affinity. The ITC experiments indicate a stoichiometry of two GAGs per KC or MIP2 dimer and that the enthalpic and entropic contributions vary significantly between the two chemokine-heparin complexes. NMR-based structural models of heparin-KC and heparin-MIP2 complexes reveal that different combinations of residues from the N-loop, 40s turn,  3-strand, and C-terminal helix form a binding surface within a monomer and that both conserved residues and residues unique to a particular chemokine mediate the binding interactions. MD simulations indicate significant residue-specific differences in their contribution to binding and affinity for a given chemokine and between chemokines. On the basis of our observations that KC and MIP2 bind to GAG via distinct molecular interactions, we propose that the differences in these GAG interactions lead to differences in neutrophil recruitment and play nonoverlapping roles in resolution of inflammation. Chemokines, a large family of small molecular weight proteins, play crucial roles in the pathophysiology of infectious and inflammatory diseases (1-3). Common to these diverse func-This work was supported by National Institutes of Health Grants P01 HL107152, R21AI124681, and S10OD023576. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This article contains Figs. S1-S3 and Movies S1 and S2.