On investigating the role of granulocyte-macrophage colony-stimulating factor (GM-CSF) and costimulatory molecule, B70, in antitumor immunity, we have found important effects of GM-CSF/B70 coexpression in the interaction with natural killer (NK) cells. We used the pLSN vector system to contain the neomycin-resistant gene and LTR promoter. The pLSNGM-CSF, pLSNB70 and pLSNB70/GM-CSF, pLSN vectors each containing GM-CSF, B70, and B70/GM-CSF cDNA, respectively, were constructed. They were transfected into human hepatocellular carcinoma cell (SK-HEP1), and stable cells (SK-pLSN, SK-GM, SK-B70 and SK-BG) were selected after neomycin treatment. According to enzyme-linked immunosorbent analysis and FACS, we showed that expression of GM-CSF was increased up to 23-fold in SK-GM and SK-BG cells, and also expression of B70 was induced at least 76-97% in SK-B70 and SK-BG cells. Expression of B70 was remarkably increased by autocrine effect of GM-CSF in SK-BG cells. Primary cytolytic ability of GM-CSF and B70 significantly increased almost 4-fold (effector/target ratio, 100:1) in SK-BG cells. In in vivo studies, SK-BG cells showed much less subcutaneous tumor formation in nude mice accompanying increased NK cell proliferation and cytotoxicity. Therefore, these results suggest that combining expression of GM-CSF and B70 may enhance NK-mediated cytotoxicity, and then induce the antitumor immunity in hepatoma transplanted into nude mice.