Better-folding Green Fluorescent Protein (GFP) mutants selected from bacterial screenings are commonly used in widely different cellular environments. However, it is unclear if the folding ef®ciency of GFPs is invariant in different cell types. In this work, we have analysed the folding properties of GFP variants in bacteria versus mammalian cells. Remarkably, S65T was found to fold at comparable levels with the wild type GFP in bacteria, but at 10-fold lower levels in mammalian cells. On the other hand, Bex1 folded 3±4 times better than the wtGFP or S65T in E. coli, and 10±20-fold or more than 95-fold better, respectively, in mammalian cells. The Vex1 mutant demonstrated similar properties to Bex1. No evidence of differential GFP unfolding in vivo or of preferential degradation of unfolded GFP molecules was found. Moreover, no relationship between GFP folding ef®ciency and expression levels, or protein stability was detected. Trivial Aconfounding factors, like GFP unfolding caused by different pH or ¯uorescence quenching due to molecular crowding, were also excluded. In summary, our results demonstrate that speci®c GFP variants follow different folding trajectories in mammalian versus bacterial cells. The speci®city of this differential folding supports a role of chaperones in guiding the folding of GFP in vivo.