Hepatic uptake and gene expression mechanisms following intravenous administration of naked plasmid DNA (pDNA) by conventional and hydrodynamics-based procedures were studied in mice. After conventional (normal) intravenous injection, ³²P-labeled pDNA was rapidly eliminated from the circulation and predominantly taken up by the liver nonparenchymal cells while no significant gene expression was observed in this organ. The hepatic uptake process was saturable. Involvement of a specific mechanism was demonstrated since the hepatic uptake of [³²P]pDNA was dramatically inhibited by cold pDNA, calf thymus DNA, and some polyanions [polyinosinic acid (poly I), dextran sulfate], but not by others (polycytidylic acid, chondroitin sulfate). The liver endothelial cells appeared to be a major contributor because gadolinium chloride (GdCl₃)-induced Kupffer cell blockade did not affect the hepatic uptake. After intravenous injection of naked pDNA with a large volume of saline at a high velocity (hydrodynamics-based procedure), the apparent hepatic uptake profile was similar to that after normal injection. The hepatic uptake was not inhibited by prior administration of polyanions, including poly I, dextran sulfate, and heparin. The hydrodynamics-based procedure resulted in marked gene expression in the liver, which was not inhibited by prior administration of polyanions or GdCl₃ treatment. These results indicate that pDNA uptake is a nonspecific process. This hypothesis was supported by the finding that significant hepatic uptake of bovine serum albumin and immunoglobulin G was observed after the hydrodynamics-based procedure.