Endothelial (eNOS) and neuronal (nNOS) nitric oxide synthase are implicated as important contributors to cerebral vascular regulation through nitric oxide (NO). However, direct in vivo measurements of NO in the brain have not been used to dissect their relative roles, particularly as related to oxygenation of brain tissue. We found in vivo rat cerebral arterioles had increased [NO] and diameter at reduced periarteriolar oxygen tension (PO₂) when either bath oxygen tension or arterial pressure was decreased. Using these protocols with highly selective blockade of nNOS, we tested the hypothesis that brain tissue nNOS could donate NO to the arterioles at rest and during periods of reduced perivascular oxygen tension, such as during hypotension or reduced local availability of oxygen. The decline in periarteriolar PO₂ by bath manipulation increased [NO] and vessel diameter comparable to responses at similarly decreased PO₂ during hypotension. To determine if the nNOS provided much of the vascular wall NO, nNOS was locally suppressed with the highly selective inhibitor N-[(4S)-4-Amino-5-[(2-aminoethyl](aminopentyl]-N'-nitroguanidine. After blockade, resting [NO], PO₂, and diameters decreased and the increase in [NO] during reduced PO₂ or hypotension was completely absent. However, flow mediated dilation during occlusion of a collateral arteriole did remain intact after nNOS blockade and the vessel wall [NO] increased ~80% of normal. Therefore, nNOS predominately increased NO during decreased periarteriolar oxygen tension, such as that during hypotension, but eNOS was the dominate source of NO for flow shear mechanisms.