The kidney importantly contributes to the long-term control of arterial pressure. In contrast to commonly used model organisms data on the function of human intrarenal arteries in particular their distal branches are scarce. Therefore we investigated agonist-induced vasoconstriction and endothelium-dependent vasodilation with particular focus on the role of the NADPH oxidase (NOX) for vascular superoxide formation and the modulation of agonist-induced constriction as well as of endothelium-dependent dilation by superoxide in distal human intrarenal arteries. Interlobar, arcuate and interlobular artery segments were dissected from tumor free parts of kidneys from patients who were nephrectomized because of a renal tumor. Sequential inhibition of oxygen radical forming systems revealed that 75% of the renal arterial superoxide formation was NOX-dependent. NOX activity was two-fold higher in distal than in proximal artery segments. The mRNA expression of the isoenzymes NOX2 and NOX4 and of the small NOX subunits p22phox and p47phox was similar in interlobar, arcuate and interlobular arteries. Phenylephrine (PE) and endothelin-1 (ET-1) induced similar maximum tension relative to depolarisation-induced tension in arcuate and interlobular arteries (pD2 6.25 0.25 and 8.22 0.07, respectively). This was not affected by the superoxide scavenger tiron. Vasopressin (AVP) was a less potent vasoconstrictor than PE and ET-1 under in vitro conditions and evidence for V1-receptor desensitization was obtained. When precontracted with 3 mmol/l PE, acetylcholine (Ach) concentration-dependently reduced arcuate and interlobular artery tone to 40% of precontraction values. Tiron significantly blunted maximum acetylcholine-induced vasodilation in both artery segments.We conclude that in vitro human intrarenal arteries show a similar responsiveness to PE, ET-1 and Ach but not to AVP as commonly used model organisms. NOX activity is the major source of superoxide in these vessels which contributes to endothelium-dependent vasodilation.