Resistance to small-molecule CCR5 inhibitors arises when HIV-1 variants acquire the ability to use inhibitor-bound CCR5 while still recognizing free CCR5. Two isolates, CC101.19 and D1/85.16, became resistant via four substitutions in the gp120 V3 region and three in the gp41 fusion peptide (FP), respectively. The binding characteristics of a panel of monoclonal antibodies (MAbs) imply that several antigenic forms of CCR5 are expressed at different levels on the surfaces of U87-CD4-CCR5 cells and primary CD4؉ T cells, in a cell-typedependent manner. CCR5 binding and HIV-1 infection inhibition experiments suggest that the two CCR5 inhibitor-resistant viruses altered their interactions with CCR5 in different ways. As a result, both mutants became generally more sensitive to inhibition by CCR5 MAbs, and the FP mutant is specifically sensitive to a MAb that stains discrete cell surface clusters of CCR5 that may correspond to lipid rafts. We conclude that some MAbs detect different antigenic forms of CCR5 and that inhibitor-sensitive and -resistant viruses can use these CCR5 forms differently for entry in the presence or absence of CCR5 inhibitors.The small-molecule CCR5 inhibitors maraviroc (MVC) and vicriviroc (VVC) are, or have been, used to treat human immunodeficiency virus type 1 (HIV-1) infection. They bind in the transmembrane helices and stabilize CCR5 in a conformation the viral Env complex cannot use efficiently (14,26,47). Resistant viruses usually gain the ability to enter cells via inhibitor-bound CCR5 while retaining the use of free CCR5 (46, 57). Virus-CCR5 binding involves interactions between the Tyr-sulfated N terminus (NT) and the second extracellular loop (ECL2) of the coreceptor and the 4-stranded bridging sheet and V3 region of the gp120 glycoprotein, respectively (20,21). In the most common genetic route to resistance, multiple sequence changes in V3 make the virus more dependent on the CCR5 NT (4,7,27,(37)(38)(39)55). A much rarer pathway involves changes in the fusion peptide (FP) of the gp41 protein, but the resistance mechanism is unknown (3). These pathways were followed when resistant isolates CC101.19 and D1/85.16 were derived from CC1/85 under selection by two similar inhibitors, AD101 and VVC, in peripheral blood mononuclear cells (PBMCs); the most critical resistance-associated substitutions in the escape mutant viruses were four in V3 and three in the FP (27,33). In this study, we used infectious Env chimeric clones, Res-4V3 derived from CC101.19 and Res-3FP from D1/85.16, together with the parental clones Par-4V3 and Par-3FP, derived from CC1/85, which were chosen based on sequence similarities with Res-4V3 and Res-3FP (7).The HIV-1 coreceptors CCR5 and CXCR4 exist in heterogeneous forms (6, 29), influenced by factors such as posttranslational modifications, coupling to G proteins, and the lipid environment (5,8,15,34,35). CCR5 monoclonal antibodies (MAbs) can vary considerably in how they stain different cell types in a way that is not always explained by CCR5 expression levels (1...