Inducible nitric oxide synthase (iNOS) catalyzes the reaction that converts the substrates O(2) and l-arginine to the products nitric oxide (NO) and l-citrulline. Macrophages, and many other cell types, upregulate and express iNOS primarily in response to inflammatory stimuli. Physiological and pathophysiological oxygen tension can regulate NO production by iNOS at multiple levels, including transcriptional, translational, posttranslational, enzyme dimerization, cofactor availability, and substrate dependence. Cell culture techniques that emphasize control of cellular PO(2), and measurement of NO or its stable products, have been used by several investigators for in vitro study of the O(2) dependence of NO production at one or more of these levels. In most cell types, prior or concurrent exposure to cytokines or other inflammatory stimuli is required for the upregulation of iNOS mRNA and protein by hypoxia. Important transcription factors that target the iNOS promoter in hypoxia include hypoxia-inducible factor 1 and/or nuclear factor κB. In contrast to the upregulation of iNOS by hypoxia, in most cell types NO production is reduced by hypoxia. Recent work suggests a prominent role for O(2) substrate dependence in the short-term regulation of iNOS-mediated NO production.