Reactive oxygen species (ROS) play important roles in renal vasoconstrictor responses to acute and chronic stimulation by angiotension II and norepinephrine, as well as in long-term effects of endothelin-1 (ET-1). Little is known about participation of ROS in acute vasoconstriction produced by ET-1. We tested the influence of NAD(P)H oxidase inhibition by apocynin (4 mg/kg/min, infused into the renal artery (ira)) on ET_A and ET_B receptor signaling in the renal microcirculation. Both receptors were stimulated by ET-1, ET_A receptors by ET-1 during ET_B antagonist BQ-788, and ET_B by ET_B agonist sarafotoxin 6C. ET-1 (1.5 pmol injected ira) reduced renal blood flow (RBF) 17±4%. Apocynin raised baseline RBF (+10±1%, p<0.001) and attenuated the ET-1 response to 10±2%, i.e., 35±9% inhibition (p<0.05). Apocynin reduced ET_A-induced vasoconstriction by 42±12% (p<0.05) and that of ET_B-stimulation by 50±8% (p<0.001). During nitric oxide (NO) synthase inhibition (LNAME), apocynin blunted ET_A-mediated vasoconstriction by 60±8% (p<0.01), whereas its effect on the ET_B-response (by 87±8%, p<0.001) was even larger without than with NO present (p<0.05). The cell-permeable superoxide dismutase mimetic tempol (5 mg/kg/min ira), which reduces O₂^- and may elevate H₂O₂, attenuated ET-1 responses similar to apocynin (by 38±6%, p<0.01). We conclude that ROS, O₂^- rather than H₂O₂, contribute substantially to acute renal vasoconstriction elicited by both ET_A and ET_B receptors and to basal renal vasomotor tone in vivo. This physiological constrictor action of ROS does not depend on scavenging of NO. In contrast, scavenging of O₂^- by NO seems to be more important during ET_B stimulation.