Reactivation of CMV can cause severe disease after allogeneic hemopoietic stem cell transplantation. Adoptive T cell therapy was successfully used for patients who had received transplants from CMV-positive donors. However, patients with transplants from CMV-negative donors are at highest risk, and an adoptive therapy is missing because CMV-specific T cells are not available from such donors. To address this problem, we used retroviral transfer of CMV-specific TCR genes. We generated CMV-specific T cell clones of several HLA restrictions recognizing the endogenously processed Ag pp65. The genes of four TCRs were cloned and transferred to primary T cells from CMV-negative donors. These CMV-TCR-transgenic T cells displayed a broad spectrum of important effector functions (secretion of IFN-gamma and IL-2, cytotoxicity, proliferation) in response to endogenously processed pp65 and could be enriched and expanded by strictly Ag-specific stimulation. Expansion of engineered T cells was accompanied by an increase in specific effector functions, indicating that the transferred specificity is stable and fully functional. Hence, we expect these CMV-TCR-transgenic T cells to be effective in controlling acute CMV disease and establishing an antiviral memory.