The loss of renal function continues to be a frequent complication of the iodinated contrast agents used to perform diagnostic angiography and endovascular procedures. This study examined the hypothesis that contrast-induced renal injury is partly due to a decrease in cortical and medullary microvascular blood flow after the downregulation of endogenous renal cortical and medullary nitric oxide (NO) synthesis. Anesthetized male Sprague-Dawley rats (300 g) had microdialysis probes or laser Doppler fibers inserted into the renal cortex to a depth of 2 mm and into the renal medulla to a depth of 4 mm. Laser Doppler blood flow was continuously monitored, and the microdialysis probes were connected to a syringe pump and perfused in vivo at 3 μL/min with lactated Ringer’s solution. Dialysate fluid was collected at time zero (basal) and 60 minutes after infusion of either saline or Conray 400 (6 mL/kg). Both groups were treated with saline carrier, N ω-nitro-L-arginine methyl ester hydrochloride (L-NAME, 30 mg/kg), L-arginine (400 mg/kg), or superoxide dismutase (10,000 U/kg), an oxygen-derived free radical scavenger. Dialysate was analyzed for total NO and eicosanoid synthesis. The renal cortex and medulla were analyzed for inducible NO synthase (iNOS), cyclooxygenase-2 (COX2), prostacyclin synthase, and prostaglandin E 2 (PGE 2) synthase content by Western blot analysis. Conray caused a marked decrease in cortical and medullary blood flow with a concomitant decrease in endogenous cortical NO, PGE 2, and medullary NO synthesis. The addition of L-NAME to the Conray further decreased cortical and medullary blood flow and NO synthesis, which were restored toward control by L-arginine. Neither L-NAME nor L-arginine (added to the Conray) altered cortical or medullary eicosanoids release. Medullary PGE 2 synthesis decreased when superoxide dismutase was added to the Conray treatment, suggesting that oxygen-derived free radicals had a protective role in maintaining endogenous medullary PGE 2 synthesis after Conray treatment. Conray did not significantly alter iNOS, COX-2, prostacyclin synthase, or PGE 2 synthase content. These findings suggest that the downregulation of renal cortical and medullary NO synthesis contributes to the contrast-induced loss of renal cortical and medullary microvascular blood flow. Preservation of normal levels of renal cortical and medullary NO synthesis may help prevent or lessen contrast-induced renal vasoconstriction and lessen contrast-induced renal injury found after diagnostic and therapeutic endovascular procedures. This study suggests that clinically relevant cortical and medullary vasodilators (NO and vasodilator prostanoids) are required to maintain microvascular renal cortical and medullary blood flow. This study combines in vivo techniques of microdialysis and laser Doppler flow probes to show that endogenous NO synthesis is important in serving a protective role to prevent vasoconstriction at the local cortical and medullary levels after radiocontrast administration. Understanding the mechanisms of contrast-induced nephropathy will help develop treatment regimens to either minimize or eliminate this important complication of contrast agents.