We previously reported that the recombinant adenovirus (Ad) vector containing the thymidine kinase (TK) gene driven by the osteocalcin (OC) promoter (Ad-OC-TK), when delivered concurrently with acyclovir (ACV), is highly selective in blocking the growth of osteosarcoma in experimental models (Cancer Res. 1996;56:4614-4619). To investigate the possible additive effects of the combined treatment of gene therapy and conventional chemotherapy (chemogene therapy), we compared the effect of low dose (IC10) methotrexate (MTX) and OC promoter-based toxic gene therapy with either of these single modalities alone. We choose low dose MTX with the intent of determining whether chemosensitization of the osteosarcoma may be possible in combination with gene therapy with an overall reduced toxicity profile and enhanced therapeutic efficacy when compared to a single agent alone. In vitro, the combined treatments of MTX (3 ng/mL) and Ad-OC-TK (20 multiplicity of infection (MOI)/target cell) plus ACV (10 mg/mL) had an additive therapeutic effect over that of either MTX (P < 0.05) or Ad-OC-TK plus ACV treatment alone (P < 0.05). In vivo, nude mice with subcutaneous tumors of either human osteosarcoma (MG-63) or rat osteosarcoma (ROS) received three intratumoral injections of Ad-OC-TK (5 x 10(8) PFU) plus daily intraperitoneal ACV (40 mg/kg body weight) for 2 weeks in combination with five weekly bolus intraperitoneal MTX (87.5 mg/kg). Osteosarcoma tumor growth was inhibited more efficiently than by either Ad-OC-TK plus ACV (P < 0.05) or MTX treatment (P < 0.005) alone. At day 45 in the ROS group, 100% of the animals survived when treated with chemogene therapy, whereas 80% survived with gene therapy and no animals survived in the MTX-treated or untreated controls. In summary, we developed a novel therapeutic strategy for the treatment of osteosarcoma employing both chemotherapy and gene therapy. Chemogene therapy could potentially achieve better antitumor effects with reduced toxicity than the conventional chemotherapy or gene therapy protocols alone.