Relapse is more frequent after autologous than allogeneic bone marrow transplantation (BMT), due in part to lack of T-lymphocyte mediated allogeneic graft-versus-leukemia (GVL) effects. Infusions of leukemia-reactive T cells to patients after autologous BMT may be a means for providing a GVL effect. Costimulation of T cells by binding of the CD28 receptor on T cells with B7-counter receptors on antigen presenting cells amplifies antigen-specific T-cell responses. To enhance generation of leukemia reactive cytotoxic T lymphocytes (CTL), the murine B7-1– and B7-2–costimulatory molecule cDNAs were introduced into the MHC class I⁺, class II⁻, murine meyloid leukemia cell line C1498. B7-1 expression greatly enhanced the ability of the leukemia cells to generate and expand leukemia reactive CTL in vitro. A highly cytolytic and C1498 specific CD8⁺ CTL line was generated by B7-1 costimulation. This CTL line proliferated autonomously and produced interleukin-2 when provided B7-1 or B7-2 costimulation by C1498 leukemia cells. To test the in vivo antileukemia properties of this CTL line, irradiated syngeneic BMT recipients were given graded doses of leukemia cells on day 0, followed by CTL infusions beginning on day 1 post-BMT. Recipients of 10⁷ CTL had a 3 log reduction in leukemia burden such that 100% of mice were protected from a supralethal leukemic cell dose. Sustained immune responses were detectable up to 3 months postinfusion of the CTL line. B7-1 or B7-2 costimulation in vivo did not augment antileukemia effects of infused CTL post BMT. These results suggest that B7 costimulation of leukemia reactive CTL may be important for their ex vivo generation and expansion for use in human adoptive immunotherapy of leukemia.