The chemokine receptor CCR5 is the principal coreceptor for R5 (macrophage-tropic) strains of HIV-1. CCR5 uses G-proteins as transducing elements. Here we report the biochemical consequences of the interaction between CCR5 and G-proteins. Macrophage inflammatory protein-1 beta (MIP-1 beta) binding to CCR5 was potently and specifically inhibited by guanine nucleotides. The molecular mechanism of this inhibitory effect was shown to be a dose-dependent reduction in MIP-1 beta receptors. We also show that the MIP-1 beta binding site is allosterically regulated by monovalent cations and that binding of this endogenous agonist is highly temperature sensitive and dependent on divalent cations, characteristic of a G-protein-coupled receptor (GPCR). HIV-1 envelope glycoprotein decreased the affinity of CCR5 for MIP-1 beta but also altered the kinetics of MIP-1 beta binding to CCR5, proving that it interacts with a distinct, but allosterically coupled binding site. The findings described herein contribute to our understanding of how CCR5 interacts with chemokines and HIV-1 envelope.