Blockade with B7 antagonists interferes with CD28:B7 and CTLA-4:B7 interactions, which may have opposing effects. We have examined the roles of CD28:B7 and CTLA-4:B7 on in vivo alloresponses. A critical role of B7:CD28 was demonstrated by markedly compromised expansion of CD28-deficient T cells and diminished graft-versus-host disease lethality of limited numbers of purified CD4+ or CD8+ T cells. When high numbers of T cells were infused, the requirement for CD28:B7 interaction was lessened. In lethally irradiated recipients, anti-CTLA-4 mAb enhanced in vivo donor T cell expansion, but did not affect, on a per cell basis, anti-host proliferative or CTL responses of donor T cells. Graft-versus-host lethality was accelerated by anti-CTLA-4 mAb infusion given early post-bone marrow transplantation (BMT), mostly in a CD28-dependent fashion. Donor T cells obtained from anti-CTLA-4 mAb-treated recipients were skewed toward a Th2 phenotype. Enhanced T cell expansion in mAb-treated recipients was strikingly advantageous in the graft-versus-leukemia effects of delayed donor lymphocyte infusion. In two different systems, anti-CTLA-4 mAb enhanced the rejection of allogeneic T cell-depleted marrow infused into sublethally irradiated recipients. We conclude that blockade of the selective CD28-B7 interactions early post-BMT, which preserve CTLA-4:B7 interactions, would be preferable to blocking both pathways. For later post-BMT, the selective blockade of CTLA-4:B7 interactions provides a potent and previously unidentified means for augmenting the GVL effect of delayed donor lymphocyte infusion.