Hydrodynamic gene delivery to the liver is a promising approach for liver gene therapy in the clinic, but levels of gene expression in larger species have been much less than in rodents. The development of surgical techniques for pressurizing individual liver segments and the establishment of whether hepatic vascular anatomy in fact permits pressurization of individual segments are critical issues that need to be addressed. We have evaluated these issues using hydrodynamic delivery to individual segments of the pig liver, via branches of both portal and hepatic veins. Our objective was to develop surgical techniques that achieve elevated vascular pressures within individual liver segments with small volumes, but without interruption of portal blood flow or reduction in venous return to the heart. We report that, without specific surgical interventions to obstruct outflow of DNA solution from the targeted liver segment, little or no increase in intrahepatic vascular pressure occurs. We demonstrate, for the first time, that selective pressurization of individual liver segments is possible without compromising portal venous flow or venous return to the heart. Thus, hydrodynamic gene delivery to individual liver segments is technically achievable in a clinical setting, but will require open abdominal surgery rather than minimally invasive techniques.