Ultrasound (US) was applied to a targeted canine liver lobe simultaneously with injection of plasmid DNA (pDNA)/microbubble (MB) complexes into a portal vein (PV) segmental branch and occlusion of the inferior vena cava (IVC) to facilitate DNA uptake. By using a 1.1 MHz, 13 mm diameter transducer, a fivefold increase in luciferase activity was obtained at 3.3 MPa peak negative pressure (PNP) in the treated lobe. For more effective treatment of large tissue volumes in canines, a planar unfocused transducer with a large effective beam diameter (52 mm) was specifically constructed. Its apodized dual element configuration greatly reduced the near-field transaxial pressure variations, resulting in a remarkably uniform field of US exposure for the treated tissues. Together with a 15 kW capacity US amplifier, a 692-fold increase of gene expression was achieved at 2.7 MPa. Transaminase and histology analysis indicated minimal tissue damage. These experiments represent an important developmental step toward US-mediated gene delivery in large animals and clinics.