Oxygen homeostasis in cells and tissues is tightly controlled by the transcription factors Hypoxia Inducible Factors (HIFs) which regulate the expression of genes that facilitate O2 delivery and metabolic adaptation to O2 deprivation in all mammalian cells (Semenza, 2001). Therefore, HIFs ensure the cells’ survival by playing a dominant role in response to ischemia, anemia or systemic hypoxia (Berchner-Pfannschmidt et al., 2008). Three isoforms of HIF-α subunits HIF-1α, HIF-2α and HIF-3α differ by their specific sources; however they share highly similar structures (Gu et al., 1998). Because of the frequent presence of HIF-1α and HIF-2α, as well the less well understood role of HIF-3α (Loboda et al., 2010), the focus of this study was HIF-1α and HIF-2α. Controlling the expression of several hundreds of HIF target genes involved in O2 transport (erythropoietin), angiogenesis, glucose metabolism, proliferation and survival (Rankin et al., 2008), HIF-1α and HIF-2α were intensively studied in recent years. However, neither the physiology nor the pathophysiology is fully resolved as “master regulators of oxygen homeostasis” (Semenza, 1998). To obtain better understanding of the HIFs’ regulation, confocal microscopic fluorescence resonance energy transfer (FRET) was applied for investigating the protein-protein interaction between the subunits. In order to show the function of basic helix-loop-helix (bHLH) and Period/Arnt/Single-minded (PAS) domains, deletion and single nucleotide mutations were performed on HIF-1α and HIF-2α. It is evident that a basis to explain some of the target gene specificity observed with HIF-1 versus HIF-2 driven genes can be provided by the new findings in this study.