Reactive oxygen species (ROS) have long been considered only as cyto- and genotoxic. However, there is now compelling evidence that ROS also act as second messengers in response to various stimuli, such as growth factors, hormones and cytokines. The hypoxia-inducible transcription factor (HIF) is a master regulator of oxygen-sensitive gene expression. More recently, HIF has also been shown to respond to non-hypoxic stimuli. Interestingly, recent reports indicate that ROS regulate HIF stability and transcriptional activity in well-oxygenated cells, as well as under hypoxic conditions. Consequently, ROS appear to be key players in regulating HIF-dependent pathways under both normal and pathological circumstances. This review summarizes the current understanding of the role of ROS in the regulation of the mammalian HIF system.