The present study was undertaken to compare the effects of a type-specific (HIV-1 MN) anti-V3 antibody on in vitro human immunodeficiency virus (HIV) infection of peripheral blood mononuclear cells in systems of cell-free versus cell-to-cell transmission of virus. Anti-V3 antibody completely prevented HIV-1 infection when cell-free virus was the sole mechanism of infection. A significant reduction of the neutralizing activity of the anti-V3 antibody was observed when infectivity was dependent on both cell-free and cell-to-cell mechanisms of infection. Furthermore, when cell-to-cell transfer of virions was the primary mechanism of HIV-1 infection, inhibition of HIV-1 infection was not observed. Therefore, a potent neutralizing antibody with a single epitope specificity failed to effectively control dissemination of a persistent HIV-1 infection in a system characterized predominantly by cell-to-cell transfer of virus.