The oxygen requirement for chromophore formation potentially limits the use of green fluorescent protein as a reporter under hypoxic conditions. In the light of this, the applicability of a hypoxia-responsive enhanced green fluorescent protein (EGFP)-based system to the measurement of tumor hypoxia was tested in human HT 1080 fibrosarcoma cells stably transfected with a destabilized EGFP vector containing the hypoxia-responsive 5HRE-hCMVmp promoter or, as a positive control, the strong constitutive CMV promoter. After various schedules of hypoxia and reoxygenation, EGFP fluorescence of live cells was assessed by flow cytometry, and protein levels were analyzed by Western blot. Fluorescence of CMV promoter positive control cells dropped to 38+/-5% of aerobic levels after 12 hours at <0.02% oxygen, but was unaffected by higher oxygen concentrations. Following 12 hours at <0.02% oxygen, cells transfected with the hypoxia-responsive vector exhibited maximum fluorescence after 4 hours of subsequent reoxygenation, reaching 68+/-2% of the levels in CMV promoter controls under aerobic conditions. With such reoxygenation, these cells exhibited a constant increase in fluorescence between 2% and <0.02% oxygen. EGFP chromophore formation is only affected by near-anoxic oxygen concentrations. The correlation of fluorescence and oxygen concentration is restored by a 4-hour reoxygenation period due to oxidation of pre-synthesized EGFP and a delayed increase in EGFP protein synthesis.