Nitric oxide (NO) production by inducible NO synthase (iNOS) is dependent on O₂ availability. The duration and degree of hypoxia that limit NO production are poorly defined in cultured cells. To investigate short-term O₂-mediated regulation of NO production, we used a novel forced convection cell culture system to rapidly (response time of 1.6 s) and accurately (±1 Torr) deliver specific O₂ tensions (from <1 to 157 Torr) directly to a monolayer of LPS- and IFNγ-stimulated RAW 264.7 cells while simultaneously measuring NO production via an electrochemical probe. Decreased O₂ availability rapidly (≤30 s) and reversibly decreased NO production with an apparent K_mO₂ of 22 (SD 6) Torr (31 μM) and a V_max of 4.9 (SD 0.4) nmol·min⁻¹·10⁻⁶ cells. To explore potential mechanisms of decreased NO production during hypoxia, we investigated O₂-dependent changes in iNOS protein concentration, iNOS dimerization, and cellular NO consumption. iNOS protein concentration was not affected (P = 0.895). iNOS dimerization appeared to be biphasic [6 Torr (P = 0.008) and 157 Torr (P = 0.258) >36 Torr], but it did not predict NO production. NO consumption was minimal at high O₂ and NO tensions and negligible at low O₂ and NO tensions. These results are consistent with O₂ substrate limitation as a regulatory mechanism during brief hypoxic exposure. The rapid and reversible effects of physiological and pathophysiological O₂ tensions suggest that O₂ tension has the potential to regulate NO production in vivo.