Endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) contributes to hepatic vascular homeostasis. The aim of this study was to examine whether delivery of an adenoviral vector encoding eNOS gene to liver affects vasomotor function in vivo and the mechanism of NO production in vitro. Rats were administered adenoviruses encoding beta-galactosidase (AdCMVLacZ) or eNOS (AdCMVeNOS) via tail vein injection and studied 1 wk later. In animals transduced with AdCMVLacZ, beta-galactosidase activity was increased in the liver, most prominently in hepatocytes. In AdCMVeNOS-transduced animals, eNOS protein levels and catalytic activity were significantly increased. Overexpression of eNOS diminished baseline perfusion pressure and constriction in response to the alpha(1)-agonist methoxamine in the perfused liver. Transduction of cultured hepatocytes with AdCMVeNOS resulted in the targeting of recombinant eNOS to a perinuclear distribution and binding with the NOS-activating protein heat shock protein 90. These events were associated with increased ionomycin-stimulated NO release. In summary, this is the first study to demonstrate successful delivery of the recombinant eNOS gene to liver in vivo and in vitro with ensuing NO production.