During low O₂ (hypoxia), HIF- is stabilized and translocates to the nucleus where it regulates genes critical for survival and/or adaptation in low O₂. While it appears that mitochondria play a critical role in HIF induction, controversy surrounds the underlying mechanism(s). To address this, we monitored HIF-2 expression and oxygen consumption in an O₂-sensitive immortalized rat adrenomedullary chromaffin (MAH) cell line. Hypoxia (2- 8% O₂) caused a concentration- and time-dependent increase in HIF-2 induction which was blocked in MAH cells with either RNAi knock-down of the Riske Fe-S-protein (RISP), a component of complex III, or knock-down of cytochrome c oxidase (COX10) subunit of complex IV, or defective mitochondrial DNA (0 cells). Additionally, pharmacological inhibitors of mitochondrial complexes I, III, IV, i.e. rotenone (1 µM), myxothiazol (1 µM), antimycin A (1 µ/ml) and cyanide (1 mM), blocked HIF-2 induction in control MAH cells. Interestingly, the inhibitory effects of the mitochondrial inhibitors were dependent on O₂ concentration such that at moderate-to-severe hypoxia (6% O₂) HIF-2 induction was blocked by low inhibitor concentrations that were ineffective at more severe hypoxia (2% O₂). Manipulation of the levels of reactive oxygen species (ROS) had no effect on HIF-2 inducation. These data suggest that in this O₂-sensitive cell line mitochondrial O₂ consumption, rather than changes in ROS, regulates HIF-2 during hypoxia.