CD8(+) memory T cells endanger allograft survival by causing acute and chronic rejection and prevent tolerance induction. We explored the role of CD27:CD70 T-cell costimulatory pathway in alloreactive CD8(+)/CD4(+) T-cell activation. CD27-deficient (CD27(-/-)) and wild-type (WT) B6 mice rejected BALB/c cardiac allografts at similar tempo, with or without depletion of CD4(+) or CD8(+) T cells, suggesting that CD27 is not essential during primary T-cell alloimmune responses. To dissect the role of CD27 in primed effector and memory alloreactive T cells, CD27(-/-) or WT mice were challenged with BALB/c hearts either 10 or 40 days after sensitization with donor-type skin grafts. Compared to WT controls, allograft survival was prolonged in day 40- but not day 10-sensitized CD27(-/-) recipients. Improved allograft survival was accompanied by diminished secondary responsiveness of memory CD8(+) T cells, which resulted from deficiency in memory formation rather than their lack of secondary expansion. Chronic allograft vasculopathy and fibrosis were diminished in CD27(-/-) recipients of class I- but not class II-mismatched hearts as compared to WT controls. These data establish a novel role for CD27 as an important costimulatory molecule for alloreactive CD8(+) memory T cells in acute and chronic allograft rejection.