Approximately 50% of the forearm vasodilatation to intra-arterial infusions of acetylcholine is mediated by endothelium-derived nitric oxide. These conclusions have been derived from venous occlusion plethysmographic measurements of total forearm blood flow during co-infusions of acetylcholine and NG-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide synthase. Since venous occlusion plethysmography measures total limb blood flow, the relative proportion of the measurement from skin cannot be determined precisely. To determine the effects of acetylcholine on skin specifically, we have used laser Doppler flowmetry to measure vascular responses to local iontophoresis of acetylcholine in the forearm of normal male volunteers. To elucidate the possible mechanisms of cutaneous vasodilatation to acetylcholine, vascular responses were measured before and after systemic inhibition of prostanoid production and nitric oxide synthesis by oral aspirin (600 mg daily for 3 days) and intravenous L-NMMA (3 mg/kg for 60 min), respectively. After aspirin administration, dose-dependent vascular responses to acetylcholine were reduced significantly by approximately 53% (p < 0.005, ANOVA). In contrast, intravenous L-NMMA appeared to have no significant effect on cutaneous vascular responses to acetylcholine. While the role of nitric oxide is uncertain, vasodilatation to acetylcholine in the forearm skin is mediated largely by a prostanoid-dependent mechanism. Assessment of cutaneous vascular responses to iontophoresis of acetylcholine may, therefore, be useful in diseases where abnormal endothelium-dependent prostanoid function has been implicated.