To improve the transduction and distribution of adenovirus in a tumor mass, we generated an adenovirus to selectively replicate in tumors. We hypothesized that after infection the replicating adenovirus would spread throughout the tumor mass and cause direct oncolysis of tumor cells. E2F transcription factors are critical regulators of cell growth and are often overexpressed in cancer cells because of the frequent aberrations in the pRb/E2F/p16(INK4a) pathway. As a result, a majority of tumor cells exist in a high proliferative state. E2F-1 is a transcription factor that activates its own transcription and that of other genes involved in the G(1) to S transition phase of the cell cycle. We constructed an adenovirus (Ad(E2F-1(RC)) so that E1A expression and viral replication were under the control of the human E2F-1 promoter element. AdE2F-1(RC) virus replicated as efficiently as the wild-type adenovirus and caused extensive cell killing in a panel of tumor cells in vitro. In contrast, nonproliferating normal epithelial, fibroblast, and endothelial cells, which express no E2F-1, were not able to support AdE2F-1(RC) replication. In animal studies, different dosing regimens of AdE2F-1(RC) administered to flank xenografts of ovarian and lung cancers led to a significant therapeutic advantage often surpassing that seen in animals treated with the wild-type adenovirus. This novel selectively replicating adenovirus offers a promising treatment platform for a variety of cancers of which the hallmark is uncontrolled cell growth.