Assembly of the Hypoxia-Inducible Factor-1 (HIF-1) complex by its oxygen-regulated subunit HIF-1a and its constitutive b-subunit is the key event of the cellular genetic response to hypoxia. To localize HIF-1a within the nucleus of hypoxic cells we applied 2-photon-laser microscopy (2PLM). 2PLM is inherently confocal and avoids the disturbance of the cellular redox status through photo reduction of oxygen, which will interfere with oxygen sensing and HIF-1a accumulation. HIF-1 assembly was studied in living cells in a specialized hypoxic chamber mounted on the microscopic stage which allows the in vivo analysis under a well defined oxygen tension. Mobility studies of fluorescently labelled HIF-1 subunits by fluorescence recovery after photo bleaching (FRAP) revealed that HIF-1a migrates more slowly than HIF-1b within the nuclear compartment. In addition, we analysed the assembly of the HIF-1 complex by fluorescence resonance energy transfer (FRET) and determined the distance of a-and b-subunits of the transcriptionally active HIF-1 complex bound to DNA. Interestingly, the N- and C-terminus of HIF-1a and -b closely co-localized. This organization of the HIF complex is different from so far described two dimensional models but is in line with the very recent structural consideration that heterodimer formation of the HIF complex relies on anti-parallel association of the PAS B domains within the a- and b-subunit.