We evaluated efficacy, toxicity and potential synergism of adenoviral-mediated thymidine kinase (tk)- ganciclovir (GCV) gene therapy in combination with 4 cytotoxic chemotherapeutic agents (doxorubicin, cisplatin, mitomycin C, and methotrexate) in 3 human bladder cancer cell lines. Cell lines were exposed to (1) 10 different concentrations of adenovirus expressing tk plus GCV; (2) 8 different concentrations of either doxorubicin, methotrexate, mitomycin C or cisplatin; or (3) combination treatment consisting of either low-, medium- or high-dose tk-GCV gene therapy plus 8 different concentrations of a single chemotherapeutic agent. Cell survival was determined using a MTT-based cell proliferation-assay. For most combinations, adding chemotherapy to tk-GCV gene therapy did not result in any therapeutic benefit. In some scenarios, we observed modest improvement with combinations of high-dose tk-GCV gene therapy and high-dose standard chemotherapy over tk-GCV monotherapy. Low concentrations of methotrexate enhanced the antitumor effects of low- and medium-dose tk-GCV gene therapy. Low level negative interference between tk-GCV gene therapy and chemotherapy occurred in some combinations but was overall negligible. In general, adding chemotherapy to tk-GCV gene therapy did not demonstrate significant therapeutic benefit in vitro. High doses of chemotherapeutic agents should be used in combination with tk-GCV gene therapy in order to take advantage of the occasional instance where modest improvement occurred with combination therapy. Additional studies exploring the role of methotrexate in enhancing the tk-GCV system are required. Investigation of other, potentially more synergistic chemotherapeutic agents in combination with tk-GCV is warranted.