Aims This study examined the interaction between reactive oxygen species and nitric oxide (NO) in mediating the decrease in renal blood flow (RBF) evoked by sympathetic renal nerve stimulation (RNS). Methods Groups of male Wistar rats were subjected to RNS at different frequencies prior to, and following, an infusion of: (i) tempol, the superoxide dismutase (SOD) mimetic, (ii) tempol plus the hydrogen peroxide‐degrading enzyme catalase (tem + cat), (iii) diethyldithiocarbamic acid (DETC), a SOD inhibitor, (iv) the nitric oxide synthase (NOS) inhibitor, L‐nitro‐arginine methyl ester (L‐NAME) alone, or (v) L‐NAME followed by tempol, into the kidney cortico‐medullary border (CMB). Blood perfusion within the cortical (CBP) and medullary (MBP) regions of the kidney was measured using Laser‐Doppler flowmetry. Results Infusion of tempol CMB significantly attenuated RNS‐evoked reductions in CBP (by 22% at 8 Hz; P < 0.05), but not MBP. When tempol and catalase were co‐infused to reduce both ROS and hydrogen peroxide (H2O2), respectively, there was a significantly greater attenuation of the RNS‐evoked reduction in CBP compared with that of tempol alone. Infusion of either DETC or L‐NAME alone did not significantly affect the CBP or MBP responses to RNS. Similarly, RNS following tempol infusion with L‐NAME also had no effect on CBP and MBP over and above the group that received tempol alone. Conclusion These results suggest that reactive oxygen species such as superoxide and H2O2 have a direct role in reducing renal vascular compliance in response to RNS, rather than indirectly through scavenging NO.