CD81 is expressed on human T cells at all stages of development. CD81 is physically associated with CD4 and CD8 and antibodies against CD81 generate signals which influence thymocyte adhesion and proliferation. Here we evaluate the function of CD81 on mature T cells. We employ a system in which B cells present superantigen to autologous T cells and find that anti-CD81 promotes T cell-B cell collaboration. Anti-CD81 induces T cell-B cell adhesion of peripheral blood lymphocytes which is partially mediated by LFA-1. CD81 engagement promotes LFA-1-dependent T cell activation, IL-2 production and proliferation. The antibody 5A6 was uniquely potent in exerting these effects compared to another antibody to CD81 or to antibodies that react with other tetraspanins expressed on T cells, anti-CD53 or anti-CD82. CD81-derived signals rapidly induce high-avidity LFA-1 as measured by cell binding to recombinant ICAM-3-coated fluorescent microspheres or by cell adhesion to ICAM-3-coated plastic. 5A6 activation of LFA-1 does not expose the high-affinity conformation epitope recognized by monoclonal antibody 24.