Endothelium-derived hyperpolarizing factor (EDHF) contributes to endothelium-dependent relaxation of isolated arteries, but it is not known whether this also occurs in the case of humans in vivo. The present study examined the role of EDHF in human forearm circulation. Forearm blood flow (FBF) was measured by strain-gauge plethysmography in 31 healthy, normal subjects (mean±SE age, 23±2 years; 24 men and 7 women). After oral administration of aspirin (486 mg), we infused N^G-monomethyl-l-arginine (8 μmol/min for 5 minutes) into the brachial artery. We used tetraethylammonium chloride (TEA, 1 mg/min for 20 minutes), a K_Ca channel blocker, as an EDHF inhibitor, and nicorandil as a direct K⁺ channel opener. TEA significantly reduced FBF (P<0.05) but did not change systemic arterial blood pressure. Furthermore, TEA significantly inhibited the FBF increase in response to substance P (0.8, 1.6, 3.2, and 6.4 ng/min, n=8) and bradykinin (12.5, 25, 50, and 100 ng/min, n=8; both P<0.001), whereas it did not affect the FBF increase in response to acetylcholine (4, 8, 16, and 32 μg/min, n=8), sodium nitroprusside (0.4, 0.8, 1.6, and 3.2 μg/min, n=8), or nicorandil (0.128, 0.256, 0.512, and 1.024 mg/min, n=8). These results suggest that EDHF contributes substantially to basal forearm vascular resistance, as well as to forearm vasodilatation evoked by substance P and bradykinin in humans in vivo.