Bicyclams are a novel class of antiviral compounds that are highly potent and selective inhibitors of the replication of HIV-1 and HIV-2. Surprisingly, however, when the prototype compound AMD3100 was tested against M-tropic virus strains such as BaL, ADA, JR-CSF, and SF-162 in human peripheral blood mononuclear cells, the compound was completely inactive. Because of the specific and potent inhibitory effect of AMD3100 on T-tropic viruses, but not M-tropic viruses, it was verified that AMD3100 interacts with the CXC-chemokine receptor CXCR4, the main coreceptor used by T-tropic viruses. AMD3100 dose dependently inhibited the binding of a specific CXCR4 monoclonal antibody to SUP-T1 cells as measured by flow cytometry. It did not inhibit the binding of the biotinylated CC-chemokine macrophage inflammatory protein (MIP) 1alpha or MIP-1beta, ligands for the chemokine receptor CCR5 (the main coreceptor for M-tropic viruses). In addition, AMD3100 completely blocked (a) the Ca2+ flux at 100 ng/ml in lymphocytic SUP-T1 and monocytic THP-1 cells, and (b) the chemotactic responses of THP-1 cells induced by stromal cell-derived factor 1alpha, the natural ligand for CXCR4. Finally, AMD3100 had no effect on the Ca2+ flux induced by the CC-chemokines MIP-1alpha, regulated on activation normal T cell expressed and secreted (RANTES; also a ligand for CCR5), or monocyte chemoattractant protein 3 (a ligand for CCR1 and CCR2b), nor was it able to induce Ca2+ fluxes by itself. The bicyclams are, to our knowledge, the first low molecular weight anti-HIV agents shown to act as potent and selective CXCR4 antagonists.