Intravenous injection of immunodeficient mice with adenoviral vectors carrying the HSV-tk gene led to preferential transduction of liver tissue. Subsequent application of ganciclovir (GCV) resulted in extremely high toxicity with negligible survival rates. This toxicity was only seen when GCV was applied in addition to the adenoviral vector and not if combined with the Ad-beta gal control vector. This indicates a specific Ad-tk/GCV-related toxicity. Low survival rates were seen not only after intravenous administration but also after injection of the vectors into the portal vein, the liver tissue and liver tumors, but not during the treatment of subcutaneous tumors. This, together with extensive signs of liver degeneration occurring as early as one day after the initiation of GCV treatment, strongly suggests a specific liver-associated toxicity. Severe toxicity was observed when animals received GCV treatment as late as 7 weeks after vector administration. Moreover, in vitro survival rates of resting primary hepatocytes treated with Ad-tk and GCV were very low. We therefore suppose a mechanism of toxicity of phosphorylated GCV which is independent from cellular proliferation. Since our results indicate that the Ad-HSV-tk/GCV approach is toxic for the resting liver, additional safety features such as tumor-restricted transgene expression should be included in adenoviral vectors.