Replication-competent adenoviral vectors hold the promise to be more efficient gene delivery vehicles than their replication-deficient counterparts, but they are also associated with a higher risk for adverse effects, especially in light of the fact that there is no established effective therapy for serious, disseminated adenovirus infection. To assess whether the therapeutic options to inhibit adenoviral replication can be enhanced by expressing a suicide gene, we examined the antiadenoviral effects of 15 drugs against wild-type adenovirus type 5 (Ad5) and an Ad5-based replication-competent vector expressing herpes simplex virus-1 thymidine kinase (HSV-tk) (Ad.OW34) using a real-time polymerase chain reaction -based assay and flow cytometry. Ad5 and Ad.OW34 were highly susceptible to the fluorinated pyrimidine analogs 5-fluoro-2'-deoxyuridine (FUdR), 5-fluorouridine (FUR), and trifluorothymidine (TFT), with a mean 50% inhibitory concentration (IC(50)) ranging from 0.12 to 0.32 microM. The mean IC(50) of ribavirin and cidofovir (CDV) for Ad5, the most frequently used drugs to treat adenovirus disease, was 6.87 and 3.19 microM, respectively. In contrast to Ad5, the Ad.OW34 vector was susceptible to (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdU, IC(50) 0.09 microM), ganciclovir (GCV, IC(50) 0.19 microM), and acyclovir (ACV, IC(50) 32.04 microM). Additionally, we demonstrated in an animal model that Ad.OW34 vector replication can be inhibited significantly by GCV, CDV, and TFT by 35.2, 7.7, and 3.7-fold, respectively, compared to untreated animals. The observed antiadenoviral effects were primarily not through cell killing, since the in vitro 50% cytotoxic concentrations (CC(50)) were more than 1000 times higher than the antiadenoviral IC(50) of the drugs examined, even in cells stably expressing HSV-tk. Since for HSV-tk-dependent inhibition of adenoviral vectors, stability of HSV-tk expression is crucial, we examined Ad.OW34 vector stability, by passaging the vector 10 times serially in the presence of 10 microM GCV. The HSV-tk/GCV system neither changed the susceptibility of Ad.OW34 to GCV significantly nor detectable vector rearrangements occurred, suggesting that the system might be suitable as a fail-safe mechanism to stop adenoviral vector replication.