Treatment options for disseminated cervical cancer remain inadequate. Here, we investigated a strategy featuring Ad5-Δ24RGD, an oncolytic adenovirus replication-competent selectively in cells defective in the Rb-p16 pathway, such as most cervical cancer cells. The viral fiber contains an α vβ3 and αvβ5 integrin-binding RGD-4C motif, allowing coxsackie-adenovirus receptor-independent infection. These integrins have been reported to be frequently upregulated in cervical cancer. Oncolysis of cervical cancer cells was similar to a wild-type control in vitro. In an animal model of cervical cancer, the therapeutic efficacy of Ad5-Δ24RGD could be demonstrated for both intratumoral and intravenous application routes. Biodistribution was determined following intravenous administration to mice. Further preclinical safety data were obtained by demonstrating lack of replication of the agent in human peripheral blood mononuclear cells. These results suggest that Ad5-Δ24RGD could be useful for local or systemic treatment of cervical cancer in patients with disease resistant to currently available modalities. © 2004 Wiley-Liss, Inc.