Gene therapy could potentially revolutionize the treatment of gastrointestinal (GI) tract cancer. The aim of this study was to establish a practical method of gene transfer which would be applicable to human gastric cancer. Retrovirus or/and adenovirus vectors carrying the lacZ marker gene were transferred in situ by needle through an endoscopic biopsy channel into primary gastric cancer in six male beagle dogs that had been treated with N-ethyl-N′-nitro-N-nitrosoguanidine (ENNG). In addition, an adenovirus vector carrying the herpes simplex virus thymidine kinase (Ad.CAGHSV-TK) gene was introduced in situ into cancer tissues in the stomach of three dog, and the animals were treated with intravenous ganciclovir (GCV). Retrovirus-producing cells which expressed the lacZ gene were specifically localized to the injection site in the stomach. The lacZ gene was more widely transferred into the tumor by the adenovirus vector than by retrovirus-producing cells. Improvement of the needle used for gene transfer and the use of multiple injections per tumor led to more diffuse transfer of the vector into the tumor. The Ad.CAG lacZ gene was also transferred into regional lymph nodes of the stomach. Moderate to diffuse degeneration of the primary cancer tissues of the stomach was found after Ad.CAGHSV-TK/GCV gene therapy. Moreover, almost complete tissue degeneration was observed in the regional lymph nodes of the stomach. An adverse effect of HSV-TK/GCV gene therapy was acute hepatotoxicity, which was not found after Ad.CAG lacZ gene transfer, but was found after high-titer Ad.CAGHSV-TK gene transfer followed by GCV. These findings suggest that in situ gene transfer of a suicide gene followed by prodrug treatment may be applicable not only to primary tumors, but also to lymph node metastases of gastric cancer, though further study of both beneficial and adverse effects is required before clinical usage.