Tissue-specific promoter has been used for cancer-specific suicide gene therapy, but its transcriptional activity is relatively low. For more efficient gene therapy of hepatocellular carcinoma, a simultaneous infection method of two recombinant adenoviruses was developed, in which one carried Cre gene under the control of alpha-fetoprotein promoter and the other a potent expression unit activated by Cre. When the vectors with lacZ reporter gene were introduced systematically into mouse models of disseminated tumors, specific and enhanced gene expression was observed exclusively in hepatocellular carcinomas both in the liver and in the lung. Next, using herpes simplex virus thymidine kinase, the anti-tumor effect was examined. Although in cultured cells, 60-300-fold expression of enzymatic activity and enhanced ganciclovir sensitivity was obtained compared with that of the single recombinant adenovirus directly driven by alpha-fetoprotein promoter, there was no significant anti-tumor effect for subcutaneous tumor on athymic mice. The lack of anti-tumor effect in mice could be explained by insufficient simultaneous transduction of the two vectors in the tumors, since it was found that a high multiplicity of infection was required to activate this system. Some strategies to overcome this dose limitation are needed, at least in the case of hepatocellular carcinoma.