Objective The V3 loop of the HIV-1 envelope glycoprotein gp120 is an important determinant of HIV–specific cell tropism. Nine different purified envelope proteins were prepared in order to examine the association between the structure of the gp20 proteins and functional properties of HIV-1 virions differing in their tropism for T-cell lines and macrophages. Results Six monoclonal antibodies to the V3 loop reacted preferentially with T-cell line-tropic gp120 envelope proteins, and one monoclonal antibody reacted preferentially with macrophage-tropic gp120 envelope proteins. T-cell line-tropic gp120 envelope proteins were at least 10-fold more susceptible to V3 loop proteolytic cleavage by human thrombin, and 1000-fold more susceptible to V3 loop proteolytic cleavage by human mast cell tryptase than macrophage-tropic gp20 envelope proteins. Conclusions These findings suggest that there are two distinct conformations for the V3 loop of T-cell line-tropic and macrophage-tropic gp20 envelope proteins.