Chronic rejection is a T cell-dependent process and blockade of the CD28-B7 T-cell costimulatory activation pathway by the fusion protein CTLA4Ig has been shown to prevent the development of accelerated graft arteriosclerosis in a rat model of chronic cardiac allograft rejection. The effectiveness of T-cell costimulatory blockade at preventing chronic allograft rejection in a clinically relevant model in combination with cyclosporine therapy has not been investigated. Using the well-established LEW into F334 heterotopic cardiac allograft model, we show that when cyclosporine is administered in combination with CTLA4Ig, it abrogates the previously demonstrated protective effect of CTLA4Ig in preventing chronic allograft rejection. Long-term surviving allografts from animals treated with a combination of cyclosporine and CTLA4Ig had a mean vascular luminal occlusion of 42.2%, affecting more than 90% of graft vessels due to accelerated arteriosclerosis. This was associated with up-regulation of intragraft expression of mRNA for CD4, the costimulatory molecule B7, the T-cell cytokine interferon-γ, monocyte chemoattractant protein-1, and the fibrogenic growth factor transforming growth factor-β; all have been previously shown to be associated with development of chronic rejection in this model. In comparison, the addition of donor splenocytes to the combination of CTLA4Ig and cyclosporine therapy protocol significantly reduced the amount of arteriosclerosis; mean vascular luminal occlusion was 11.3%, affecting approximately 50% of vessels. This was associated with decreased intragraft expression of CD4, B7, interferon-γ, monocyte chemoattractant protein-1, and transforming growth factor-β. These data indicate that the mechanism of action of CTLA4Ig in attenuating chronic rejection is cyclosporine sensitive, and that strategies implying combination of CTLA4Ig and cyclosporine may not be clinically desirable. Administration of donor antigen may be necessary if CTLA4Ig and cyclosporine are to be combined, to prevent the process of chronic rejection.