The cytomegalovirus(CMV) promoter is considered one of the strongest positive regulators. In this study toxicity, cell killing efficacy and bystander effect of Rous Sarcoma Virus(RSV) driven herpes simplex thymidine kinase(TK) gene therapy was compared with CMV driven TK gene therapy in three ovarian cancer cell lines with different growth patterns using a 3-(4,5-dimethylthiazol)-2,5-diphenyl tetra-zolium bromide (MTT) based assay. ADV/CMV-TK was shown to be 2 to 10 times more effective in tumor cell killing than ADV/RSV-TK. The difference in cell killing efficacy between ADV/CMV-TK and ADV/RSV-TK was dependent on the individual cell line. A CMV promoter dependent eight to ten fold improvement in cell killing efficacy was observed in the relatively slow growing SKOV3 cell line which is not easily transducible, while only a 2 to 4 fold difference was observed in the easily transducible OV-CA-2774 and OV-CA-1225 cell lines. ADV/CMV-TK also showed a stronger bystander effect than ADV/RSV-TK in all three ovarian cancer cell lines. Our data demonstrated that the efficacy of adenovirus-mediated gene therapy of ovarian cancer can be enhanced by using the CMV promoter without increasing toxicity.