Objective: The V3 loop of the HIV-1 envelope glycoprotein gp120 is an important determinant of HIV-1-specific cell tropism. Nine different purified envelope proteins were prepared in order to examine the association between the structure of the gp120 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 gp120 envelope proteins. Conclusions: These findings suggest that there are two distinct conformations for the V3 loop of T-cell line-tropic and macrophage-tropic gp120 envelope proteins.