Memory T cells have specific properties that are beneficial for rapid and efficient protection from pathogens previously encountered by a host. These same features of memory T cells may be deleterious in the context of a transplanted organ. Consistent with this contention is the accumulating evidence in experimental transplantation that previously sensitized animals are resistant to the effects of costimulatory blockade. Using a model of murine cardiac transplantation, we now demonstrate that alloreactive memory CD4(+) T cells prevent long-term allograft survival induced through donor-specific cell transfusion in combination with anti-CD40 ligand Ab (DST/anti-CD40L). We show that memory donor-reactive CD4(+) T cells responding through the direct or indirect pathways of allorecognition provide help for the induction of antidonor CD8(+) T effector cells and for Ab isotype switching, despite DST/anti-CD40L. The induced pathogenic antidonor immunity functions in multiple ways to subsequently mediate graft destruction. Our findings show that the varied functions of alloreactive memory CD4(+) T cells remain intact despite DST/anti-CD40L-based costimulatory blockade, a finding that will likely have important implications for designing approaches to induce tolerance in human transplant recipients.