Sodium cotransporters have a substantial role in neuronal damage during brain hypoxia. We proposed these cotransporters have beneficial roles in oxygen sensing mechanisms that increase periarteriolar nitric oxide ([NO]) during mild to moderate oxygen deprivation. Our prior studies have shown that cerebral nNOS is essential for [NO] responses to decreased oxygen tension and eNOS is of little consequence. In this study, we explored the mechanisms of three specific cotransporters known to play a role in the hypoxic state; KB-R7943 for blockade of the Na⁺/Ca²⁺ exchanger, bumetanide for the Na⁺-K⁺-2Cl^- cotransporter, and amiloride for the Na⁺/H⁺ cotransporters. In vivo measurement of arteriolar diameter and [NO] at normal and locally reduced oxygen tension in the in vivo rat parietal cortex provided the functional analysis. As previously found for intestinal arterioles, bumetanide sensitive cotransporters are primarily responsible for sensing reduced oxygen because the increased [NO] and dilation were suppressed. The Na⁺/Ca²⁺ exchanger facilitated increased nitric oxide formation because blockade also suppressed [NO] and dilatory responses to decreased oxygen. Amiloride sensitive Na⁺/H⁺ cotransporters did not significantly contribute to the microvascular regulation. To confirm that nNOS rather than eNOS was primarily responsible for NO generation, eNOS was suppressed with the fusion protein cavtratin for the caveolae domain of eNOS. Although the resting [NO] decreased and arterioles constricted as eNOS was suppressed, most of the increased NO and dilatory response to oxygen were preserved because nNOS was functional. Therefore, nNOS activation secondarily to Na⁺-K⁺-2Cl^- cotransporter and Na⁺/Ca²⁺ exchanger functions are key to cerebral vascular oxygen responses.