Endothelial-derived nitric oxide (NO) is classically viewed as a regulator of vasomotor tone. NO plays an important role in regulating O₂ delivery through paracrine control of vasomotor tone locally and cardiovascular and respiratory responses centrally. Very soon after the cloning and functional characterization of the endothelial nitric oxide synthase (eNOS), studies on the interaction between O₂ and NO made the paradoxical finding that hypoxia led to decreases in eNOS expression and function. Why would decreases in O₂ content in tissues elicit a loss of a potent endothelial-derived vasodilator? We now know that restricting our view of NO as a regulator of vasomotor tone or blood pressure limited deeper levels of mechanistic insight. Exciting new studies indicate that functional interactions between NO and O₂ exhibit profound complexity and are relevant to diseases states, especially those associated with hypoxia in tissues. NOS isoforms catalytically require O₂. Hypoxia regulates steady-state expression of the mRNA and protein abundance of the NOS enzymes. Animals genetically deficient in NOS isoforms have perturbations in their ability to adapt to changes in O₂ supply or demand. Most interestingly, the intracellular pathways for O₂ sensing that evolved to ensure an appropriate balance of O₂ delivery and utilization intersect with NO signaling networks. Recent studies demonstrate that hypoxia-inducible factor (HIF) stabilization and transcriptional activity is achieved through two parallel pathways: (1) a decrease in O₂-dependent prolyl hydroxylation of HIF and (2) S-nitrosylation of HIF pathway components. Recent findings support a role for S-nitrosothiols as hypoxia-mimetics in certain biological and/or disease settings, such as living at high altitude, exposure to small molecules that can bind NO, or anemia.