Arachidonic acid is metabolized by the 15-lipoxygenase-1 pathway to the vasodilatory eicosanoids hydroxy-epoxyeicosatrienoic acid and trihydroxyeicosatrienoic acid. We determined the in vitro and in vivo effects of the 15-lipoxygenase-1 metabolites in rabbits infected with adenovirus containing cDNA for human 15-lipoxygenase-1 (Ad-15-LO-1). Forty hours after intravenous adenoviral injection, 15-lipoxygenase-1 expression increased in liver and mesenteric arteries 10-fold and 3-fold, respectively. Expression of 15-LO-1 was limited to the endothelium of mesenteric arteries. Overexpression did not occur in tissues from rabbits infected with a beta-galactosidase containing adenovirus. Trihydroxyeicosatrienoic acid and hydroxy-epoxyeicosatrienoic acid synthesis per milligram of tissue increased by 2.1- and 1.5-fold, respectively, in mesenteric arteries from Ad-15-LO-1-infected rabbits compared with normal rabbits. Pretreatment with a 15-lipoxygenase inhibitor BW755C inhibited the synthesis. NO and prostaglandin-independent, maximal relaxations to acetylcholine were greater in mesenteric arteries from Ad-15-LO-1-infected rabbits (98.3+/-1.7%) compared with normal (60.93+/-10.5%) and beta-galactosidase containing adenovirus-infected rabbits (68.3+/-7.7%). Pretreatment with BW755C decreased these relaxations. Mean arterial pressure and heart rate did not differ in Ad-15-LO-1-infected rabbits compared with normal or beta-galactosidase containing adenovirus-infected rabbits. The hypotensive responses to acetylcholine in the presence and absence of inhibition of NO and prostaglandins were greater in Ad-15-LO-1-infected rabbits (-52+/-2% and -47+/-2%) compared with normal (-31+/-3% and -25+/-5%) or beta-galactosidase containing adenovirus-infected rabbits (-38+/-2% and -30+/-3%). Therefore, increased expression of 15-LO-1 increases acetylcholine relaxation in arteries and hypotensive responses in rabbits because of increased hydroxy-epoxyeicosatrienoic acid and trihydroxyeicosatrienoic acid synthesis.