Although many factors are thought to contribute to the regulation of metabolic vasodilation in skeletal muscle vasculature, recent interest has focused on the role of the endothelium. We examined the relative roles of nitric oxide (NO) and of vasodilator prostanoids in the control of metabolically induced functional hyperemia in the forearm of humans. In 43 healthy volunteers [24 +/- 5 (SD) yr] we assessed resting and functional hyperemic blood flow (FHBF) in response to 2 min of isotonic forearm exercise before and after inhibition of NO and/or vasodilator prostanoid production with intra-arterial NG-monomethyl-L-arginine (L-NMMA, 2 mg/min) and aspirin (ASA, 3 mg/min), respectively. Blood flow was measured using venous occlusion plethysmography. L-NMMA and ASA decreased resting forearm blood flow by 42% (P < 0.0001) and 23% (P < 0.0001), respectively, whereas infusion of ASA followed by L-NMMA reduced flow by a further 24% (P < 0.05). L-NMMA reduced peak FHBF by 18% [from 13.9 +/- 1.0 to 11.4 +/- 1.1 (SE) ml. 100 ml forearm-1. min-1, P = 0.003] and the volume "repaid" after 1 and 5 min by 25% (8.9 +/- 0.7 vs. 6.7 +/- 0.7 ml/100 ml, P < 0.0001) and 37% (26.6 +/- 1.8 vs. 16.8 +/- 1.6 ml/100 ml, P < 0.0001). ASA similarly reduced peak FHBF by 19% (from 14.5 +/- 1.1 to 11.8 +/- 0.9. 100 ml forearm-1. min-1, P < 0.001) and the volume repaid after 1 and 5 min by 14% (7.5 +/- 0. 6 vs. 6.4 +/- 0.6 ml/100 ml, P = 0.0001) and 20% (21.2 +/- 1.5 vs. 16.9 +/- 1.5 ml/100 ml, P < 0.0001), respectively. The coinfusion of ASA and L-NMMA did not decrease FHBF to a greater extent than either agent alone. These data suggest that endothelium-derived NO and vasodilator prostanoids contribute to resting blood flow and metabolic vasodilation in skeletal muscle vasculature in healthy humans. Although these vasodilator mechanisms operate in parallel in exercise-induced hyperemia, they appear not to be additive. Other mechanisms must also be operative in metabolic vasodilation.