The purpose of this study was to determine whether superoxide anions (O[Formula: see text]·) activate 5′-nucleotidase (5′-ND), thereby increasing the production of renal adenosine and regulating renal function. Using HPLC analysis, we found that incubation of renal tissue homogenate with the O[Formula: see text]· donor KO2doubled adenosine production and increased the maximal reaction velocity of 5′-ND from 141 to 192 nmol · min−1· mg protein−1. The O[Formula: see text]·-generating system, xanthine/xanthine oxidase increased the maximal reaction velocity of 5′-ND from 122 to 204 nmol · min−1· mg protein−1. Superoxide dismutase (SOD) with catalase produced a concentration-dependent reduction of 5′-ND activity in renal tissue homogenate, while the SOD inhibitor diethyldithiocarbamic acid significantly increased 5′-ND activity. Inhibition of disulfide bond formation by thioredoxin or thioredoxin reductase significantly decreased xanthine/xanthine oxidase-induced activation of renal 5′-ND. In in vivo experiments, inhibition of SOD by diethyldithiocarbamic acid (0.5 mg · kg−1· min−1iv) enhanced renal vasoconstriction induced by endogenously produced adenosine and increased renal tissue adenosine concentrations under control condition and in ischemia and reperfusion. We conclude that oxidative stress activates 5′-ND and increases adenosine production in the kidney and that this redox regulatory mechanism of adenosine production is important in the control of renal vascular tone and glomerular perfusion.