The cytomegalovirus (CMV) promoter is considered one of the strongest positive regulators leading to expression of higher levels of the thymidine kinase (TK) enzyme than the Rous Sarcoma virus (RSV) promoter in vitro and in vivo. Cell killing efficacy of adenovirus-mediated CMV promoter-driven herpes simplex virus (HSV) TK gene therapy has been found to be 2 to 10 times more effective than RSV driven HSV-TK gene therapy in vitro. In this study the impact of CMV- versus RSV-driven HSV-TK gene therapy on long-term survival of nude mice bearing human ovarian cancer has been evaluated using a prospective randomized experimental design. The experiment was designed to show significance of survival differences from a 50% increase of survived days at a p-value of 0.05 with a power of 80%. All treatment groups showed an increase in median survival compared with control groups. Treatment benefit was ADV/CMV-TK vector dose dependent. At a given viral dose, no significant prolongation of survival was observed comparing CMV- and RSV-driven ADV-TK indicating that simply increasing cell killing efficacy in vitro above a minimal threshold level using a stronger promoter may not lead to prolongation of survival in the HSV-TK/GCV system.