Although oxygen is required for functional chromophore formation during the maturation process of fluorescent proteins, the effects of hypoxia on their fluorescence have rarely been studied in mammalian cells. We recently reported that severe hypoxia (pO(2) < 0.1%) abrogates fluorescence from the fluorescent ubiquitination-based cell cycle indicator (Fucci) expressed in HeLa cells. Fucci is a system for visualizing cell cycle progression in live cells using red (monomeric Kusabira Orange 2, mK02) and green (monomeric Azami Green, mAG) fluorescent proteins. In this study, taking advantage of the system, we attempted to determine the dependence on oxygen tension (pO2) of these two fluorescent proteins during the maturation process. The oxygen tension at which the number of fluorescence-positive cells was reduced by 50% (pO(2).50) was 0.9% and 0.3% for mK02 and mAG, respectively. Furthermore, we measured fluorescence recovery kinetics after reoxygenation in cells treated at two different pO2 levels, and observed that mK02 exhibits slower kinetics of oxidation than mAG. Thus, we demonstrate that mK02 exhibits a stronger dependence on oxygen tension than mAG, as well as the usefulness of this novel method to produce varying levels of hypoxic conditions. (C) 2012 Elsevier Inc. All rights reserved.