Previous studies have shown adenoviral transfer of the herpes simplex virus thymidine kinase (HSVtk) gene followed by the anti-viral drug ganciclovir (GCV) can be used to successfully treat established human mesothelioma tumors growing within the peritoneal cavities of severe combined immune deficient (SCID) mice. These findings raised a number of questions important to the applicability, efficiency, and safety of this treatment strategy. In this report, we have further characterized the use of recombinant adenovirus carrying the HSVtk gene to treat mesothelioma and other localized malignancies. Our results indicate that the Ad.RSVtk/GCV system is effective in causing tumor regression in animals inoculated with another mesothelioma cell line and a lung cancer cell line and that animals with bulky disease can be successfully treated. Effects are seen at a wide range of virus doses and significant anti-tumor activity is present at doses of ganciclovir that are clinically achievable. Finally, this treatment approach appears safe, with limited dissemination of virus using a sensitive RT-PCR detection system. These studies further characterize the use of adenoviral transfer of the HSVtk gene to treat experimental mesothelioma and suggest that clinical trials using this approach may be feasible.