We report a novel method for targeting adenovirus-mediated gene delivery. By irradiating mammalian cells prior to adenoviral transduction, adenoviral gene transfer is greatly improved and the adenoviral genome integrates into cellular DNA. In this work, human and rodent cell lines were irradiated and subsequently transduced with the adenovirus vector Ad5CMVlacZ. Initial levels of transduction were as much as 40-fold higher in irradiated cells, and this improvement in transduction was radiation dose dependent. The duration of lacZ expression in irradiated cells was also much longer than in nonirradiated cells and reached a plateau after 21 days. At doses of 7 Gy, long-term (< 50 day) expression of lacZ could be detected in 15% of cells by flow cytometry. This long-lasting expression of lacZ was due to viral DNA integration into the host genome. Thus, pretreatment of cells with ionizing radiation improves both immediate transduction efficiency and duration of transgene expression. This may lead to the development of new protocols combining radiation and gene therapy in treating human malignancy.