The role of nitric oxide (NO) produced by NO synthase 1 (NOS1) in the renal vasculature remains undetermined. In the present study, we investigated the influence of systemic inhibition of NOS1 by intravenous administration of Nω-propyl-l-arginine (l-NPA; 1 mg · kg−1· h−1) and N5-(1-imino-3-butenyl)-l-ornithine (v-NIO; 1 mg · kg−1· h−1), highly selective NOS1 inhibitors, on renal cortical and medullary blood flow and interstitial NO concentration in Sprague-Dawley rats. Arterial blood pressure was significantly decreased by administration of both NOS1-selective inhibitors (−11 ± 1 mmHg with l-NPA and −7 ± 1 mmHg with v-NIO; n = 9/group). Laser-Doppler flowmetry experiments demonstrated that blood flow in the renal cortex and medulla was not significantly altered following administration of either NOS1-selective inhibitor. In contrast, the renal interstitial level of NO assessed by an in vivo microdialysis oxyhemoglobin-trapping technique was significantly decreased in both the renal cortex (by 36–42%) and medulla (by 32–40%) following administration of l-NPA ( n = 8) or v-NIO ( n = 8). Subsequent infusion of the nonspecific NOS inhibitor Nω-nitro-l-arginine methyl ester (l-NAME; 50 mg · kg−1· h−1) to rats pretreated with either of the NOS1-selective inhibitors significantly increased mean arterial pressure by 38–45 mmHg and significantly decreased cortical (25–29%) and medullary (37–43%) blood flow. In addition, l-NAME further decreased NO in the renal cortex (73–77%) and medulla (62–71%). To determine if a 40% decrease in NO could alter renal blood flow, a lower dose ofl-NAME (5 mg · kg−1· h−1; n = 8) was administered to a separate group of rats. The low dose of l-NAME reduced interstitial NO (cortex 39%, medulla 38%) and significantly decreased blood flow (cortex 23–24%, medulla 31–33%). These results suggest that NOS1 does not regulate basal blood flow in the renal cortex or medulla, despite the observation that a considerable portion of NO in the renal interstitial space appears to be produced by NOS1.