Macrophage nitric oxide (NO) production and hypoxia coexist during wound healing, and have been implicated in the pathogenesis and pathophysiology of multiple disease states including sepsis and cancer. Macrophages stimulated with pathogen associated molecular patterns (PAMPs) produce NO via inducible nitric oxide synthase (iNOS) from molecular O2, L-arginine, and NADPH. The first aim of this research was to characterize the degree and duration of hypoxia which would limit NO production by PAMPs stimulated macrophages. The second aim was to identify the contributing mechanism(s). Using a novel forced convection cell culture system, we demonstrated that NO production was rapidly (within seconds) and reversibly regulated by physiological and pathophysiological O2 tensions (pO2). The effect of pO2 on NO production was not mediated by changes in iNOS protein concentration or iNOS dimerization, implicating limitation of the reactant(s) as the predominant causative mechanism. In addition to O2 limitation, hypoxia has the potential to affect NADPH and L-arginine availability. In PAMPs stimulated macrophages, NADPH is predominantly produced by the oxidative pentose phosphate cycle (OPPC). NO production directly correlated with OPPC activity over a wide range of pO2, and inhibition of NO production with the specific iNOS inhibitor, 1400W, significantly decreased OPPC activity. OPPC activity increased significantly in response to chemically mediated oxidative stress irrespective of pO2, and NO production was unaffected by increasing cellular oxidative stress, indicating that NADPH availability for NO production was not limited by hypoxia. L-arginine is required for iNOS dimerization, and iNOS dimerization was maintained or increased during hypoxic exposure, suggesting sufficient L-arginine was available. Furthermore, the effect of L-arginine depletion on NO production was much slower than the response observed due to changes in pO2. In conclusion, decreased O2 availability is the predominant mechanism responsible for rapidly and reversibly limiting NO production by PAMPs stimulated macrophages exposed to acute hypoxia.