The three-dimensional structure of a member of the β subfamily of chemokines, human macrophage inflammatory protein-1β (hMIP-1β), has been determined with the use of solution multidimensional heteronuclear magnetic resonance spectroscopy. Human MIP-1β is a symmetric homodimer with a relative molecular mass of ∼16 kilodaltons. The structure of the hMIP-1β monomer is similar to that of the related α chemokine interleukin-8 (IL-8). However, the quaternary structures of the two proteins are entirely distinct, and the dimer interface is formed by a completely different set of residues. Whereas the IL-8 dimer is globular, the hMIP-1β dimer is elongated and cylindrical. This provides a rational explanation for the absence of cross-binding and reactivity between the α and β chemokine subfamilies. Calculation of the solvation free energies of dimerization suggests that the formation and stabilization of the two different types of dimers arise from the burial of hydrophobic residues.