Hepatocellular carcinoma is a lethal disease and methods that develop effective cellular-based immunotherapy are needed. We retrovirally transduced non-immunogenic mouse Hepa1-6 hepatoma cells with the gene encoding the membrane form of macrophage colony stimulating factor (mM-CSF). Excess recombinant M-CSF and phagocytosis-inhibiting chemicals blocked macrophage-mediated killing of cloned mM-CSF transfected Hepa1-6 hepatoma cells. Macrophages derived from Hck(-/-)Fgr(-/-) and Lyn(-/-) triple knockout mice, which are incapable of performing phagocytosis, failed to kill the mM-CSF transduced cells. The mM-CSF transfected tumor clones failed to grow when injected into C57BL/6 or C57L/J mice. Splenocytes from these vaccinated mice displayed cytotoxicity against parental Hepa1-6 cells, but not against B16 and CT-26 tumor cells in vitro. Mice that rejected the mM-CSF transfected Hepa1-6 tumor subsequently rejected parental Hepa1-6 cells but not the B16 melanoma cells when rechallenged. Elimination of the CD8+ effector cells by an anti-CD8 antibody and complement treatment prevented the adoptive transfer of anti-Hepa1-6-specific immunity into naive animals. Thus, mM-CSF provides a method of generating effective anti-tumor immune responses by macrophages and cytotoxic T cells against the parental Hepa1-6 cells. Our work suggests that mM-CSF transduced hepatoma cells could be used as a tumor vaccine to stimulate immune responses against hepatocellular carcinoma.