Recipients of allogeneic transplants are at risk of cytomegalovirus (CMV) infection and disease during the period of immune compromise after transplantation. The limitations of current antiviral pharmacotherapy have led to attempts to develop alternative strategies for preventing or treating CMV infection, such as adoptive transfer of donor-derived virus-specific T cells. Methods for generating CMV-specific T cells either use live CMV to infect autologous antigen-presenting cells (APCs) or require some knowledge of the immunodominant peptides involved in the immune response. A novel culture system was developed that does not use live virions and in which the APCs are monocyte-derived dendritic cells (DCs). APCs were pulsed with CMV antigen and cocultured with autologous peripheral blood lymphocytes from donors seropositive for CMV. The culture-output cells consisted of both CD4- and CD8-expressing T cells. Proliferation, as determined by a tritium-thymidine-incorporation assay, showed significant CMV-antigen specificity in cultures from 15 of 15 donors seropositive for CMV. In cytotoxicity assays, cytotoxic T lymphocytes from 10 of 12 cocultures specifically lysed autologous CMV-infected fibroblasts or DCs but not HLA-mismatched or uninfected target cells, and this activity was shown to be blocked by HLA class 1 blocking antibodies. T-cell-receptor spectratyping of cells from the cultures typically showed complex size-distribution patterns, with all size classes of a normal preculture distribution present. However, a few size-class peaks were expanded compared with the preculture patterns and these may have represented expansions of CMV-specific T-cell clones. Advantages of this culture system are that it requires no live virions and no detailed knowledge of the antigenic peptides involved and it is applicable to CMV-seropositive donors of any HLA type.