The human immunodeficiency virus type 1 (HIV-1) gp120 V3 loop plays a predominant role in chemokine receptor usage; however, other linear and nonlinear gp120 domains are involved in this step of the HIV-1 replication cycle. At present, the functional relationship between V3 and these domains with regard to coreceptor usage is unclear. To gain insights into the nature of this relationship in naturally selected viral variants, we developed a recombinant strategy based on two different gp120 backbones derived from CXCR4 (X4)- and CCR5 (R5)-tropic viral strains, respectively. Using this recombinant model system, we evaluated the phenotype patterns conferred to chimeric viruses by exogenous V3 loops from reference molecular clones and samples from infected subjects. In 13 of 17 recombinants (76%), a comparable phenotype was observed independently of the gp120 backbone, whereas in a minority of the recombinant viruses (4/17, 24%) viral infectivity depended on the gp120 context. No case of differential tropism using identical V3 sequence in the two gp120 contexts was observed. Site-directed mutagenesis experiments were performed to evaluate the phenotypic impact of specific V3 motifs. The data indicate that while the interaction of HIV-1 with chemokine receptors is driven by V3 loop and influenced by its evolutionary potential, the gp120 context plays a role in influencing the replication competence of the variants, suggesting that compensatory mutations occurring at sites other than V3 are necessary in some cases.