Prior studies with recombinant viruses constructed in vitro showed that the V2 domain of envelope gp120, in addition to the required V3 domain, enhances the efficiency of infection of primary macrophages by HIV-1. Present structural studies on the gp120s of these recombinant viruses using three human monoclonal antibodies directed to the V3 loop indicate that the V2 domain affects cell tropism by modulating the conformation of the V3 loop. Additional mutational analyses of the V2 domain of the T-cell line-tropic virus HIV-1SF2 reveal that single amino acid sequence changes, mainly those affecting the location of potential N-linked glycosylation sites and the positive charge of this region, can also alter tropism. These amino acid substitutions in the V2 domain, however, do not appear to alter the conformation of the V3 loop. Thus, the V2 domain of gp120 can influence cell tropism through both an effect on V3 as well as via a V3-independent mechanism.