Objective: To evaluate, in vitro, the cytotoxicity of the herpes simplex virus thymidine kinase (HSV1-tk) gene and ganciclovir (GCV) treatment, a widely used prodrug/suicide gene therapy; in human tongue cancer cell line Tbp-TL. Methods: Retrovirus expressing plasmid-liposome complex transduction was used. After treatment with G418 for 3 weeks, the integrated HSV1-tk gene was detected by PCR test with the primers specifically designed for HSV1-tk cDNA; HSV1-tk MRNA was detected with in situ hybridization method. In vitro cytotoxicity effect of HSV-GCV on Tbp-TL was detected with MTT and trypan blue exclusion test. Results: In vitro experiments demonstrated dose- and time-dependent cell death by transduction of the HSV1-tk gene followed by GCV treatment. The IC50 (the concentration requi4ed to elicit 50% growth inhibition) shifted from 1000 µg/mL in non-transduced Tbp-TL cells to 1.1 µg/mL in Tbp-TL/TK cells transduced with HSV1-tk gene, with the therapeutic index of 1000. Treatment with GCV at a dose of 10 µg/mL for 5 days led to complete cell death in HSV1-tk transduced tumor cells. When mixed with tk positive cells, the non-transduced cells (both the parent Tbp-TL cells as well as another non-transduced cell, the human muco-epidermoid carcinoma cell line M3SP2) were also killed by a degree associated with the percentage of tk positive cells. Seventy to 90% of the mixed cells were killed with only 10% to 20% tk positive cells involved. Conclusion: These results suggest that the HSV-tk/GCV approach to human tongue cancer cell line may be efficacious, with a wide therapeutic range, and that it exerts a bystander effect in vitro.