We have previously shown that costimulation blockade using a combination of monoclonal antibodies (mAbs) – CTLA4Ig, antibodies to CD154, LFA-1, and OX40L – can induce tolerance of cardiac allografts in mice with adoptively transferred CD4+ memory T cells [1]. However, the effect of costimulatory blockade in secondary allograft rejection has not been studied. B6 mice that rejected BALB/c skin grafts for more than 4 weeks (defined as alloantigen-primed mice) were used as recipients. The recipient mice were treated with the mAbs to CD154, LFA-1, OX40L, and CD122 on days 0, 2, 4, and 6 after the secondary transplantation of BALB/c heart. The mean survival time (MST) of secondary cardiac allografts in rats treated with antibodies to CD154 and LFA-1 (2-antibodies approach) and those treated with antibodies to CD154, LFA-1, OX40L, and CD122 (4-antibodies approach) was greater than that of the controls (MST = 6.7 days, 22.2 days, and 3.2 days, respectively). The 4-antibodies approach prevented lymphocytic infiltration in the grafts, inhibited memory T-cells proliferation in the spleen, increased IL-10 secretion in the serum, and enhanced the expression of CD4+ Foxp3+ regulatory T cells (Tregs) in spleen. Expression levels of alloreactive antibodies were high in the recipient mice of experimental and control groups. Inhibiting the memory T cells by costimulation blockade extended allograft survival in secondary transplant models but could not induce tolerance of graft. Alloreactive antibodies may participate in alloresponse and play an important role in secondary cardiac allograft rejection.