The vast majority of clinical protocols involving gene therapy today rely on viral vectors for gene transduction. The primary reason that plasmid vectors have not been widely used for gene therapy trials is their relatively low rate of stable gene transfer. We show here that ionizing radiation can improve plasmid transfection efficiency in both normal and neoplastic human and mouse cells. As high as 1,400-fold improvement in transfection efficiency can be seen in primary human fibroblasts treated with 9 Gy. Radiation improves transfection efficiency in a dose-dependent manner of only linearized plasmid DNA in transformed or immortalized cells, but of both linearized and supercoiled plasmid in normal human fibroblasts. The gene transfer dose-response curves are linear for neoplastic cell lines and exponential for primary cell lines. This suggests that radiation can improve gene integration by at least two mechanisms, one that may require free DNA ends and one that does not. The 2-hr delay described here, from the time of irradiation to the beginning of enhanced gene integration, suggests an inducible process that becomes active after the bulk of the radiation damage has been repaired. Our data further suggest that radiation may be useful to target human gene therapy using plasmid vectors.