A cyclic protein was produced in vivo using the intein from Pyrococcus furiosus PI-PfuI in a novel approach to create a circular permutation of the precursor protein by introducing new termini in the intein domain. Green fluorescent protein (GFP) was cyclized with this method in vivo on milligram scales. There was no by-product of linear or polymerized species isolated, unlike with other in vitro or in vivo cyclization methods utilizing inteins. Cyclized GFP unfolded at half the rate of the linear form upon chemical denaturation and required >2 days in 7 m guanidine hydrochloride until a residual fast folding phase (consistent with a persistent cis-proline) had disappeared. Cyclic GFP might become a novel tool for studying the role of termini and backbone topology in various biological processes such as protein degradation and translocation in vivo as well as in vitro.