Hypoxic response and inflammation both involve the action of the hypoxia-inducible transcription factors HIF-1 and HIF-2. Previous studies have revealed that both HIF- proteins are in a number of aspects similarly regulated post-translationally. However, the functional interrelationship of these two isoforms remains largely unclear. The polarization of macrophages controls functionally divergent processes; one of these is nitric oxide (NO) production, which in turn is controlled in part by HIF factors. We show here that the HIF- isoforms can be differentially activated: HIF-1 is induced by Th1 cytokines in M1 macrophage polarization, whereas HIF-2 is induced by Th2 cytokines during an M2 response. This differential response was most evident in polarized macrophages through HIF- isoform-specific regulation of the inducible NO synthase gene by HIF-1, and the arginase1 gene by HIF-2. In silico modeling predicted that regulation of overall NO availability is due to differential regulation of HIF-1 versus HIF-2, acting to, respectively, either increase or suppress NO synthesis. An in vivo model of endotoxin challenge confirmed this; thus, these studies reveal that the two homologous transcription factors, HIF-1 and HIF-2, can have physiologically antagonistic functions, but that their antiphase regulation allows them to coordinately regulate NO production in a cytokine-induced and transcription-dependent fashion.