Avoiding transduction of normal tissue after intravenous application of oncolytic adenoviruses (Ad) is an important strategy to improve the safety and efficacy of these vectors in gene therapy. As a model for a targeted vector, we used Ad vectors with type 35 fibers (Ad5/35), which efficiently transduce human cervical carcinoma cells but not liver cells. In an in vitro model of liver metastases, in which small nests of HeLa cells were surrounded by mouse hepatocytes, we showed that an Ad5/35-based conditionally replicating vector regulated by DNA replication-dependent recombination conferred increased gene transfer to tumor cells and enhanced viral replication and tumor cell lysis compared to the nontargeted Ad5 vector. Intravenous injection of Ad5/35 vectors into mice bearing liver metastases derived from HeLa cells caused markedly less hepatotoxicity than Ad5 vectors; however, it did not result in enhanced tumor cell transduction, viral replication, or oncolysis. Apparently, other factors, including the stability of virus in the blood, trapping within the liver sinusoids, transendothelial transfer, and/or vector diffusion of viral particles to tumor cells, limit tumor transduction, even if the vector is not taken up by liver cells.