The chemokine receptors CCR5 and CXCR4 function as coreceptors for human immunodeficiency virus (HIV) and are attractive targets for the development of anti-HIV drugs. The most potent CXCR4 antagonists described until today are the bicyclams. The prototype compound, AMD3100, exhibits potent and selective anti-HIV activity against CXCR4-using (X4) viruses and showed antiviral efficacy in X4 HIV-1-infected persons in a phase II clinical trial. However, AMD3100 lacks oral bioavailability due to its high overall positive charge. Initial structure-activity relationship studies with bicyclam analogues suggested that the bis-macrocyclic structure was a prerequisite for anti-HIV activity. Now, we report that the N-pyridinylmethylene cyclam AMD3465, which lacks the structural constraints mentioned above, fully conserves all the biological properties of AMD3100. Like AMD3100, AMD3465 blocked the cell surface binding of both CXCL12 (the natural CXCR4 ligand), and the specific anti-CXCR4 monoclonal antibody 12G5. AMD3465 dose-dependently inhibited intracellular calcium signaling, chemotaxis, CXCR4 endocytosis and mitogen-activated protein kinase phosphorylation induced by CXCL12. Compared to the bicyclam AMD3100, AMD3465 was even 10-fold more effective as a CXCR4 antagonist, while showing no interaction whatsoever with CCR5. As expected, AMD3465 proved highly potent against X4 HIV strains (IC50: 1-10 nM), but completely failed to inhibit the replication of CCR5-using (R5) viruses. In conclusion, AMD3465 is a novel, monomacrocyclic anti-HIV agent that specifically blocks the interaction of HIV gp120 with CXCR4. Although oral bioavailability is not yet achieved, the monocyclams, with their decreased molecular charge as compared to the bicyclams, embody an important step forward in the design of oral CXCR4 antagonists that can be clinically used as anti-HIV drugs.