We sought to generate alloreactive leukemia-reactive cytotoxic T lymphocytes from haploidentical parents by culture of peripheral blood mononuclear cells in vitro with irradiated leukemic blasts and IL-2 from children with ALL. After 21 days in culture the mean cytotoxicity of haploidentical lymphocytes against ALL blasts was 38% (n = 11). Cultured parental CTL were not leukemia specific but alloreactive as evidenced by equivalent cytotoxicity against stimulating ALL blasts and ConA-stimulated blasts from the other parent. The cultures yielded primarily CD8(+) T cells (59%). Irradiation of CTL limited proliferation by 96% but had no short-term effects on leukemia reactive cytotoxicity, suggesting a means to limit GVHD potential in vivo. One patient was treated for relapse of ALL post-haploidentical transplant with CTL generated from the original donor. A total of nine infusions were given: the first three were irradiated, while the last six were not due to disease progression. The patient experienced clearance of peripheral blasts, and despite concomitant infusion of IL-2 with the last three CTL infusions, did not experience immediate GVHD reactions. We conclude that ALL blasts are sufficiently immunostimulatory to generate in vitro CTL with provision of exogenous IL-2, and that these CTL could exert an anti-leukemia effect in vivo.