Hypoxia-inducible factor 1 (HIF-1) is a heterodirheric transcription factor induced in cells exposed to low oxygen. The oxygen-regulated subunit HIF-1a is stabilized in cells under hypoxic conditions. HIF-1 activates a large number of genes hat promote cell adaptive responses during hypoxia. The mechanisms that regulate expression of HIF-1a are not well understood. We provide evidence that inhibition of mitochondrial electron transport chain (ETC) activity prevents HIF-1a activation during hypoxia, and that inhibition of mitochondrial adenine nucleotide translocase (ANT) function results in HIF1a activation. The mechanisms and consequences of mitochondrial control of HIF-1a regulation will be addressed in three specific aims. (I) In the first specific aim we will use respiratory chain inhibitors to explore the mechanisms of HIF-1a activation during hypoxia. (II) In the second specific aim we will test the hypothesis that nitric oxide (NO) prevents HIF-1a hypoxic activation via primary inhibition of mitochondrial electron transport. (III) In the third specific aim we will explore the mechanisms by which inhibition of ANT leads to activation of HIF-1a. This is a novel pathway by which mitochondria participate in the regulation of HIF-1a. Excessive production of nitric oxide (NO), which can cause neuronal death by severe mitochondrial damage, is associated with brain ischemia/reperfusion injury. ANT itself is a target of ischemia/ reperfusion cell injury. It is believed that HIF-1 has a major role in mediating adaptive responses and possibly survival in oxygen-deprived cells. Elucidating the role of mitochondria in the regulation of HIF-1 activation will help to understand basis of oxygen homeostasis, and also to improve therapeutic strategies in these clinically relevant situations. Cultured cells and isolated mitochondrial preparations will be used to address specific questions. Techniques used for specific experimental purposes include: RNA and protein detection assays, DNA electrophoretic mobility shift assay, gene reporter assays, confocal microscopy and measurement of mitochondrial ROS production, cellular metabolic assays and biochemical assays of mitochondrial respiratory complexes.