Acetylcholine-induced endothelium-dependent vasodilation in conduit arteries primarily depends on nitric oxide (NO). However, the biochemical mediators in the microvasculature remain less well defined. We tested whether prostaglandins and NO are responsible for cutaneous acetylcholine-mediated vasodilation and if they interact to modulate vasodilation. We measured skin blood flow (SBF) using laser Doppler flow (LDF) with intradermal microdialysis in the calves of 23 healthy volunteers. We examined the response of SBF to different doses of acetylcholine (0.01-100 mM), the nonisoform-specific NO synthase inhibitor, nitro-L-arginine (NLA, 10 mM), the nonspecific cyclo-oxygenase (COX) inhibitor, ketorolac (Keto, 10 mM), and combined NLA + Keto. NLA had no effect on baseline SBF, while Keto increased baseline SBF by approximately 150%. The increase was blunted with combined NLA + Keto. SBF increased by approximately 700% with the highest acetylcholine concentration and reduced by approximately 60% by NLA. Ketorolac alone also reduced the response to acetylcholine, although the reduction varied between 10 and 20% at differing acetylcholine doses. NLA plus ketorolac reduced the responses to different doses of acetylcholine by some 30%, which was intermediate to NOS or COX inhibition alone. These data suggest that cutaneous acetylcholine-mediated endothelium-dependent vasodilation is highly NO-dependent and is also strongly related to the interactions of NO with prostaglandins.