The current study evaluated the role of CD40/CD40 ligand (CD40L) and CD28/B7 costimulation signals during alloimmune responses independently mediated by CD4+ or CD8+ T cells. Allogeneic hepatocytes were transplanted into CD8 or CD4 knock out (KO) mice under cover of costimulatory blockade. Rejection of FVB/N (H-2q) hepatocytes occurred by day 10 posttransplant in untreated CD8 or CD4 KO (H-2b) mice. Treatment of CD8 or CD4 KO mice with anti-CD40L monoclonal antibody (mAb; MR1) resulted in significant prolongation of hepatocyte survival indicating that CD40/CD40L interactions were critical in both CD4+ and CD8+ T-cell initiated hepatocyte rejection. Anti-CD40L mAb also prolonged hepatocyte survival in B-cell KO (H-2b) mice, indicating that the efficacy of CD40/CD40L blockade in preventing hepatocyte rejection was B-cell (and antibody) independent. In contrast, treatment with CTLA4 fusion protein (CTLA4Ig), prolonged hepatocyte survival in CD8 KO but not CD4 KO mice, showing that CD28/B7 interactions were important in CD4+ but not CD8+ T-cell initiated hepatocyte rejection. Under selected circumstances, such as in CD40 KO mice, both CD4+ and CD8+ T cells mediate hepatocyte rejection in the absence of CD40/CD40L costimulation and without a significant contribution from CD28/B7 costimulation signals. These results highlight the disparate roles of CD40/CD40L and CD28/B7 costimulation signals in CD4+ versus CD8+ T-cell mediated immune responses to allogeneic hepatocytes. The CD4+ T-cell independent, CD40L-sensitive, CD28/B7-independent pathway of CD8+ T-cell activation in response to transplantation antigens is novel.