Nearly all persons newly infected with HIV-1 harbor exclusively CCR5-using virus. CXCR4-using variants eventually arise in up to 50% of patients infected with subtypes B or D. This transition to efficient CXCR4 utilization is often co-incident with progression to AIDS. The basis for HIV-1's initial dependence on CCR5, the selective force(s) that drive CXCR4-utilization, and the evolutionary pathways by which it occurs are incompletely understood. Greater knowledge of these processes will inform interventions at all stages, from vaccination to cure. The determinants of co-receptor use map primarily, though not exclusively, to the V3 loop of gp120. In this study, we describe five clonal variants with identical V3 loops but divergent CXCR4 use. Mutagenesis revealed two residues controlling this phenotypic switch: a rare polymorphism in C1 and a highly conserved N-glycan in C2. To our knowledge, this is the first description of co-receptor usage regulated by the N-glycan at position 262.