MIP-1beta is a CC-chemokine that plays a role in inflammation and host defense mechanisms by interacting with its specific receptor CCR5. CCR5 is a major coreceptor for macrophage-tropic human immunodeficiency virus (HIV), and as a consequence, MIP-1beta can inhibit HIV entry. It is therefore of interest to understand how MIP-1beta and other CCR5 ligands bind to their receptor, as such understanding could lead to the rational design of more efficient HIV entry blockers. We have previously demonstrated the importance of Phe13, and of basic residues of the 40's loop, in mediating high-affinity binding of MIP-1beta to CCR5. We have now investigated further the relative contribution of other MIP-1beta residues in the interaction of the chemokine with CCR5, by studying the functional consequences of point mutations within the N-loop and the 3(10) turn of MIP-1beta, affecting the charge, size, and H-bonding properties of the side chains. Our data suggest that, in addition to Phe13, three amino acids of the N-loop and 3(10) turn (Arg18, Lys19, and Arg22) interact with CCR5 through their positive charge. We also found that Pro21 contributes to the CCR5 binding properties of MIP-1beta. Moreover, NMR spectroscopy has revealed that the presence of Tyr at position 15 is necessary for the proper folding of the chemokine. Our results therefore demonstrate that the binding determinants of MIP-1beta consist of residues arranged on one surface of the protein, including most of the basic residues in MIP-1beta, as well as two key hydrophobic groups. The good correlation observed between the potency of the mutants in a functional assay and their binding affinity strongly argues that basic residues Arg18, Lys19, and Arg22 of MIP-1beta are essential for its CCR5 binding properties, without a primary effect on CCR5 activation.