Arachidonic acid (AA) metabolites from the 15-lipoxygenase-1 (15-LO-1) pathway, trihydroxyeicosatrienoic acids (THETAs) and hydroxy-epoxyeicosatrienoic acids (HEETAs), are endothelium derived hyperpolarizing factors (EDHFs) and relax rabbit arteries. Rabbit vascular 15-LO-1 expression, THETA and HEETA synthesis and nitric oxide (NO) and prostaglandin (PG)-independent relaxations to acetylcholine (ACH) and AA decreased with age (neonates to16 weeks old). We characterized age-dependent acetylcholine (ACH) hypotensive responses in vivo in 1, 4, 8, and 16 weeks old rabbits and the contribution of THETAs and HEETAs to these responses. In anesthetized rabbits, blood pressure responses to ACH (4-4000 ng/kg) were determined in the presence of vehicle or various inhibitors. ACH responses decreased with age (p0.001). In the absence or presence of nitro-L-arginine methyl ester (LNA-ME) and indomethacin (INDO) maximum responses in 1- (-54.7±7.4 % and -37.9±3.9%) and 4- (-48.8±2.4% and -35.5±7.8%) week-old rabbits were higher than 8 (-30.0±2.8% and -26.6± 4.4%) and 16 (-36.7±3.5% and -27.3±10%) week-old rabbits. A lipoxygenase inhibitor, BW755C, reduced THETA and HEETA synthesis in mesenteric arteries. In presence of INDO and LNA, ACH relaxations were reduced by BW755C to a greater extent in the mesenteric arteries from the younger rabbits. In 4 week-old rabbits treated with LNAME and INDO, the maximum ACH hypotension was reduced, by potassium channel inhibitors apamin and charybdotoxin to -6.9±0.9%, by apamin alone to -19.5±1.4%, and by BW755C to -18.8±3.5%. The present study indicates that the age-related decrease in ACH-induced hypotension is mediated by the decreased synthesis of 15-LO-1 metabolites, THETAs and HEETAs.