Chemokines and their receptors play a critical role in host immune surveillance and are important mediators of human immunodeficiency virus (HIV) pathogenesis and inflammatory response. The chemokine receptors CCR5 and CXCR4, which act as co-receptors along with CD4 for HIV docking and entry, are down-modulated by their respective ligands, MIP-1β/SDF-1α or by the HIV envelope protein, gp120. We have studied the role of the proteasome pathway in the down-regulation of these receptors. Using the yeast and mammalian two-hybrid systems, we observed that the CCR5 receptor is constitutively associated with the ζ subunit of proteasome. Immunoprecipitation studies in CCR5 L1.2 cells revealed that this association was increased with MIP-1β stimulation. The proteasome inhibitors, lactacystin and epoxomicin, attenuated MIP-1β induced CCR5 down-modulation as detected by fluorescence-activated cell sorter analysis and confocal microscopy. The proteasome inhibitors also inhibited the SDF-1α and gp120 protein-induced down-modulation of the CXCR4 receptor in Jurkat cells. However, the inhibitors had no significant effect on the gp120-induced internalization of the CD4 receptor. These inhibitors also blocked cognate ligand-mediated chemotaxis but had no effect on SDF-1α-induced p44/42 MAP kinase or MIP-1β-induced p38 kinase activities, thus indicating differential effects of the inhibitors on signaling mediated by these receptors. These results indicate that the CCR5 and CXCR4 receptor down-modulation mechanism and chemotaxis mediated by these receptors are dependent upon proteasome activity.