Interleukin-8 (IL-8 or CXCL8), the archetypal member of the CXC chemokine subfamily, stimulates neutrophil chemotaxis by activating receptors CXCR1/IL8RA and CXCR2/IL8RB. Previous mutational studies have implicated both the N-terminal and third extracellular loop (E3) regions of these receptors in binding to IL-8. To investigate the interactions of these receptor elements with IL-8, we have constructed soluble proteins in which the N-terminal and E3 elements of either CXCR1 or CXCR2 are juxtaposed on a soluble scaffold protein; these are termed CROSS-N(X1)E3(X1) and CROSS-N(X2)E3(X2), respectively. Isothermal titration calorimetry and nuclear magnetic resonance spectroscopy were used to compare the IL-8 binding properties of the receptor mimics to those of control proteins containing only the N-terminal or E3 receptor element. CROSS-N(X2)E3(X2) bound to monomeric IL-8 with the same affinity and induced the same chemical shift changes as the control protein containing only the N-terminal element of CXCR2, indicating that the E3 element of CXCR2 did not contribute to IL-8 binding. In contrast, CROSS-N(X1)E3(X1) bound to IL-8 with 10-fold increased affinity and induced different chemical shift changes compared to the control protein containing only the N-terminal element of CXCR1, suggesting that the E3 region of CXCR1 was interacting with IL-8. However, a chimeric protein containing the N-terminal region of CXCR1 and the E3 region of CXCR2 (CROSS-N(X1)E3(X2)) bound to IL-8 with thermodynamic properties and induced chemical shift changes indistinguishable from those of CROSS-N(X1)E3(X1) and substantially different from those of CROSS-N(X2)E3(X2). These results indicate that the N-terminal and E3 regions of CXCR1 interact synergistically to achieve optimal binding interactions with IL-8.