In passaging experiments, we isolated HIV strains resistant to MAb3952, a chemokine (C-C motif) receptor 5 (CCR5) monoclonal antibody (MAb) that binds to the second extracellular domain (extracellular loop 2 [ECL-2]) of CCR5. MAb3952-resistant viruses remain CCR5-tropic and are cross-resistant to a second ECL-2-specific antibody. Surprisingly, MAb3952-resistant viruses were more susceptible to RoAb13, a CCR5 antibody binding to the N terminus of CCR5. Using CCR5 receptor mutants, we show that MAb3952-resistant virus strains preferentially use the N terminus of CCR5, while the wild-type viruses preferentially use ECL-2. We propose this switch in the CCR5 binding site as a novel mechanism of HIV resistance.HIV infects host cells by attaching to the CD4 receptor and subsequently binding to one of two major coreceptors, chemokine (C-C motif) receptor 5 (CCR5) or chemokine (C-X-C motif) receptor 4 (CXCR4). Both coreceptors are members of the superfamily of G-protein-coupled receptors, which are characterized by seven transmembrane helices, three extracellular loops, an amino-terminal domain (NTD), and an intracellular domain (2, 3). In CCR5, the two major binding regions for the viral gp120 envelope protein (Env) are the NTD and extracellular loop 2 (ECL-2) (8,9,26).Individuals who are homozygous for the CCR5 ⌬32 deletion have a natural resistance to HIV-1 infection, while heterozygous individuals have a reduced rate of infection; homozygous CCR5 ⌬32 individuals are healthy and immunocompetent, implying that normal CCR5 may be dispensable (7,19,27). This makes CCR5 an attractive target for antiretroviral drug development. The U.S. Food and Drug Administration approved maraviroc (Selzentry) in 2007 as the first small-molecule CCR5 inhibitor (20). Other CCR5 inhibitors, both small molecules (vicriviroc, aplaviroc, INC9B47, and others) and anti-CCR5 monoclonal antibodies (CCR5 MAb) (HGS004 and Pro-140) are or have been in clinical development (11, 16, 17, 22; E.