The fundamental basis for immunotherapy of leukemia is that leukemic cells express specific antigens that are not expressed by normal hematopoietic cells. However, the host immune system appears to be tolerant to leukemia cells. To overcome this tolerance, we vaccinated immunocompetent mice with murine leukemia cells (WEHI-3B and BCR-ABL+ 32D cells) transduced with a specifically constructed transmembrane form of granulocyte-macrophage colony-stimulating factor (tmGM-CSF). The transduced cells expressed tmGM-CSF on the cell-surface. To determine whether tmGM-CSF-expressing WEHI-3B leukemia cells would prevent leukemia formation as a vaccine, immunocompetent mice (BALB/c and C3H/HEJ) were immunized with lethally irradiated murine leukemia cells expressing cell-surface tmGM-CSF before challenging mice with murine leukemia cells. Two immunocompetent mouse models were investigated, either WEHI-3B cells in BALB/c mice or BCR-ABL+ 32D cells in C3H/HEJ mouse. The results showed that 100% of WEHI-3B/tmGM-CSF-vaccinated BALB/c mice and about 65% of 32D+ BCR-ABL/tmGM-CSF-vaccinated C3H/HEJ mice were protected from leukemia after leukemia cell challenge, whereas all non-vaccinated mice succumbed to leukemia. Spleen and marrow cell suspensions from vaccinated mice challenged with WEHI-3B cells lacked detectable GFP+ WEHI-3B cells at 82 days post-challenge. A significant delay of death was observed in C3H/HEJ mice challenged with the very aggressive DA-1 cell line expressing BCR-ABL. Vaccination of mice with WEHI-3B/CD40L cells protected 80% of the mice from the WEHI-3B challenge. Notably, 60% of the WEHI-3B/BALB/c mice were also protected from leukemia when WEHI-3B/tmGM-CSF vaccination was carried out after the leukemia challenge. In order to determine whether cellular immunity is involved in this vaccine-mediated protection, either CD4+ or CD8+ T cells were depleted from mice after the WEHI-3B/tmGM-CSF vaccination. The results indicate that CD8+ T-cells mediated the protective immune response provided by the irradiated tmGM-CSF-expressing WEHI-3B cells. In addition, vaccination of nude mice did not provide protection from WEHI-3B leukemia induction. Importantly, 80% of non-vaccinated mice were also protected from a WEHI-3B cell challenge after receiving spleen cells from vaccinated mice 1 day before challenge with leukemia cells. These results indicate that overexpression of tmGM-CSF on the leukemia cell-surface can enhance the recognition of leukemic cells by CD8+ T cells, and can either prevent or significantly delay leukemia induction. These findings suggest that injection of irradiated leukemia cells expressing cell-surface-bound GM-CSF has the potential as an immunological approach to treat leukemia.