Cytomegalovirus (CMV) infection increases the risk of complications after renal transplantation, but the mechanisms controlling donor-derived infection are not adequately characterized. Here, we assessed the risk of clinically significant CMV disease in donor-seropositive, recipient-seropositive (D+R+) renal transplantation and examined recipients' CMV antigen-specific cellular immune responses primed directly by donor cells. In a retrospective cohort of 569 patients administered standardized basiliximab-tacrolimus-mycophenolate-corticosteroid immunosuppressive therapy, CMV disease rates increased in D+R+ serostatus pairings compared with D-R+ pairings (hazard ratio [HR], 2.61; 95% confidence interval [CI], 1.36 to 5.01; P=0.004) and associated with increased donor-recipient HLA mismatch in the D+R+ group (HR [per class 1 mismatch], 1.43; 95% CI, 1.12 to 1.82]; P=0.02). D+R+ and D+R- transplants in which the donor and recipient differentially expressed at least one HLA class I allele were followed prospectively from the time of transplantation. During the first year after transplantation, four of eight seropositive recipients and one of three seronegative recipients displayed peripheral blood CD8+ T cell responses to CMV presented by recipient-specific HLA. Notably, no recipients mounted responses to CMV presented by donor-specific HLA, despite the detection of CMV antigen expression in all seropositive donor organs examined (n=10), suggesting that the allograft of Class I HLA-mismatched seropositive donors is inaccessible to CD8+ T cell responses. Finally, pretransplant assays of anti-CMV cellular immunity predicted post-transplant CMV replication less accurately in D+R+ pairings than in D-R+ pairings, possibly reflecting in vitro assay specificity for recipient, rather than donor, HLA. These findings are relevant to the clinical management and immunologic understanding of donor-transmitted viral infection.