The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several vasodilating factors, including prostacyclin, nitric oxide, and endothelium-derived hyperpolarizing factor (EDHF). We have recently identified that endothelium-derived hydrogen peroxide (H₂O₂) is an EDHF in mice. The present study was designed to examine whether this is also the case in humans. Bradykinin elicited endothelium-dependent relaxations and hyperpolarizations in the presence of indomethacin and N^ω-nitro-l -arginine, which thus were attributed to EDHF, in human mesenteric arteries. The EDHF-mediated relaxations were significantly inhibited by catalase, an enzyme that specifically decomposes H₂O₂, whereas catalase did not affect endothelium-independent hyperpolarizations to levcromakalim. Exogenous H₂O₂ elicited relaxations and hyperpolarizations in endothelium-stripped arteries. Gap junction inhibitor 18α-glycyrrhetinic acid partially inhibited, whereas inhibitors of cytochrome P450 did not affect the EDHF-mediated relaxations. These results indicate that H₂O₂ is also a primary EDHF in human mesenteric arteries with some contribution of gap junctions.