Costimulatory signals are absolutely required for T-cell activation after T-cell receptor/major histocompatibility complex-peptide interaction. So far, the best-known candidate essential costimulatory signal is mediated by interaction of CD28 on T cells with B7 on antigen- presenting cells. Using an allogeneic B7+ Epstein-Barr virus- transformed B-cell line as stimulator, we found that addition of a monoclonal antibody (MoAb) to B7 that efficiently blocks B7-CD28 interaction only partially inhibited proliferation and interleukin-2 (IL-2) production in primary and secondary mixed lymphocyte reactions (MLR), whereas the generation of cytotoxic T lymphocytes (CTL) was not affected. Inhibition of primary or secondary MLR-induced T-cell activation with cyclosporin A (CsA) at nontoxic concentrations also was never complete. However, the combination of CsA and anti-B7 MoAb B7–24 synergistically blocked allogeneic B cell-induced T-cell proliferation, IL-2 production, and CTL generation. These data suggest that the mere blockage of B7-CD28 interaction during allotransplantation will be insufficient to prevent rejection or graft-versus-host disease. However, low CsA concentrations, when combined with an agent blocking B7-CD28 interaction, can potentially achieve complete immunosuppression.