The chemokine receptors CCR5 and CXCR4, in combination with CD4, mediate cellular entry of macrophage-tropic (M-tropic) and T-cell-tropic strains of human immunodeficiency virus type 1 (HIV-1), respectively, while dualtropic viruses can use either receptor. We have constructed a panel of chimeric viruses and envelope glycoproteins in which various domains of the dualtropic HIV-1_DH12 gp160 were introduced into the genetic background of an M-tropic HIV-1 isolate, HIV-1_AD8. These constructs were employed in cell fusion and virus infectivity assays using peripheral blood mononuclear cells, MT4 T cells, primary monocyte-derived macrophages, or HOS-CD4 cell lines, expressing various chemokine receptors, to assess the contributions of different gp120 subdomains in coreceptor usage and cellular tropism. As expected, the dualtropic HIV-1_DH12gp120 utilized either CCR3, CCR5, or CXCR4, whereas HIV-1_AD8 gp120 was able to use only CCR3 or CCR5. We found that either the V1/V2 or the V3 region of HIV-1_DH12 gp120 individually conferred on HIV-1_AD8 the ability to use CXCR4, while the combination of both the V1/V2 and V3 regions increased the efficiency of CXCR4 use. In addition, while the V4 or the V5 region of HIV-1_DH12 gp120 failed to confer the capacity to utilize CXCR4 on HIV-1_AD8, these regions were required in conjunction with regions V1 to V3 of HIV-1_DH12 gp120 for efficient utilization of CXCR4. Comparison of virus infectivity analyses with various cell types and cell fusion assays revealed assay-dependent discrepancies and indicated that events occurring at the cell surface during infection are complex and cannot always be predicted by any one assay.