Our purpose was to evaluate hyporesponsivity to NO-induced dilation in small arterioles during nitrate tolerance. An Alza osmotic pump was implanted in the left flank of adult rats (n=56), for continuous administration of nitroglycerine (140µg/hr) or vehicle (propylene glycol). On post-operative day 3, arcade (~50µm diameter) and terminal arterioles (~20µm) were observed in the cremaster preparation, using in vivo video microscopy. Local vascular responses were obtained with micropipette applied NO-donors, with and without superoxide dismutase (SOD), MnTBAP, or losartan. On day 3, NO-mediated dilation was significantly attenuated in nitroglycerine treated rats. Attenuation was greater in the terminal arterioles compared to the arcades. Control responses were restored by SOD, MnTBAP, or losartan, suggesting a role for elevated angiotensin II and reactive oxygen species (ROS) as mediators of the attenuated NO-dilation (nitrate tolerance). Addition of losartan to the drinking water, likewise prevented nitrate tolerance. In summary, terminal arterioles are affected by nitrates to a greater extent than the arcade arterioles that feed them, in a process dependent on angiotensin II and ROS.