Delivery of cancer chemotherapy directly to the cancer cell has great appeal. Previous studies using adenoviral transfer of the herpes simplex virus thymidine kinase (HSV-tk) gene followed by ganciclovir (GCV) in an ascites model of breast cancer was successful in reducing tumor burden and prolonging life. However, increasing the viral dose resulted in increased toxicity and host mortality emphasizing the need for an improved therapeutic ratio. To test the hypothesis that enhancement of HSV-tk gene expression would lead to increased sensitivity to GCV and improved bystander effect, we created breast cancer cells expressing HSV-tk under the control of the inducible tetracycline promoter. Using this system, we could inducibly increase gene expression and biochemical activation of HSV-tk. These increased levels of HSV-tk decreased the IC50 to GCV nearly 50-fold. However, the bystander effect was not enhanced by increasing HSV-tk gene expression. We conclude that increased HSV-tk gene expression improves sensitivity to CCV. However, additional measures, such as increased gap junction communication, will likely be needed to enhance the bystander effect and the therapeutic efficacy of this strategy.