We investigated relationships between cytomegalovirus (CMV) seropairing and CMV prophylaxis on graft outcome in recipients of solid organ transplants. Transplants carried out from 1985 to 2002 and reported to the Collaborative Transplant Study were analyzed. In cadaver kidney recipients, CMV prophylaxis was significantly associated with improved graft survival only in the seronegative-recipient/seropositive-donor combination (at 3 years: 79.4% with prophylaxis vs. 73.5% without prophylaxis; RR 0.80, p < 0.0001). Among patients who had a functioning graft at 1 year, significantly fewer patients who received CMV prophylaxis received rejection treatment in the preceding year (26.3%), compared with patients who did not receive prophylaxis (32.4%) (p = 0.0001), suggesting an inhibitory effect of CMV prophylaxis on acute rejection. Significant improvements in graft survival after CMV prophylaxis were found also in CMV-negative recipients of CMV-positive heart, and lung or heart-lung transplants, but not liver transplants. The age of the recipient had a differential effect on graft and patient survival after CMV prophylaxis. Use of antilymphocyte antibodies or mycophenolate mofetil was not associated with an enhanced CMV effect on graft outcome. These results may contribute to a better understanding of the influence of pretransplant CMV serology on the effect of CMV prophylaxis.