Macrophage migration inhibitory factor (MIF) is a cytokine that mediates inflammatory diseases. MIF promotes atherogenic leukocyte recruitment through a promiscuous, yet highly affine, interaction with CXCR2 and CXCR4. Binding to CXCR2 is dependent on a pseudo-(E)LR motif in MIF, but a second interaction site has been elusive. Here we identified an N-like loop in MIF, suggesting that MIF binding to CXCR2 follows the 2-site binding mode of bona fide chemokines. For MIF, the model predicts interactions between the N-like loop and the CXCR2 N domain (site 1) and pseudo-(E)LR and extracellular loops (ELs) of CXCR2 (site 2). Applying biophysical and peptide array analysis, we demonstrated an interaction between MIF and the CXCR2 N domain, which was pseudo-(E)LR independent. Peptide array analysis also indicated that the pseudo-(E)LR motif is responsible for MIF binding to EL2 and 3. Notably, peptides MIF-(40-49) and MIF-(47-56), representing N-like-loop-derived peptides, but not a scrambled control peptide, significantly blocked MIF/CXCR2 binding, MIF-mediated monocyte arrest under flow on aortic endothelial cells in vitro (IC(50): 1.24×10(-6) M), and MIF-dependent monocyte adhesion to atherosclerotic mouse carotid arteries in vivo. Thus, the N-like loop in MIF is critical for MIF's noncognate interaction with CXCR2 and proatherogenic functions. The 2-site binding model that explains chemokine receptor activation also applies to MIF.