Gene therapy as a form of molecular medicine is expected to have a major impact on medical treatments in the future. However, the clinical use of gene therapy today is hampered by inadequate gene delivering systems to ensure sufficient, accurate and safe DNA uptake in the target cells in vivo. Nonviral transfection methods might have the advantage of safe application, but it would be helpful to increase their transfection rates, especially in vivo. In this study, we show that focused ultrasound provides an enhanced transfer of DNA plasmids in vitro and in vivo. In vitro, the beta-galactosidase and luciferase DNA reporter plasmid were transfected into four cell lines (NIH 3T3 fibroblasts, malignant melanoma Mewo, HeLa, Dunning prostate tumor R3327-AT1). Ultrasound induced a 55- (Mewo) to 220-fold (AT1) stimulation resulting in transfection efficiencies in vitro between 2% (Mewo) and 12% (AT1). The in vivo stimulation was assessed in the Dunning prostate tumor R3327-AT1 implanted subcutaneously in Copenhagen rats using the beta-galactosidase reporter. After intratumoral DNA injection, focused ultrasound induced a 10-fold increase of beta-galactosidase positive cells in histology and a 15-fold increase of beta-galactosidase protein expression in the ELISA assay. In contrast, ultrasound was not found to enhance reporter gene expression after intravenous plasmid application. Because ultrasound waves can be focused on different anatomical locations in the human body without significant adverse effects, the control of DNA transfer by focused ultrasound is a promising in vivo method for spatial regulation of gene-based medical treatments.