Green fluorescent proteins (GFPs) are being intensively investigated due to both their unusual optical spectroscopic characteristics and the extraordinary utility of GFPs as tools in biochemistry, cell biology, and molecular genetics. Recent studies have suggested that the spectrophotometric and fluorescence characteristics of GFPs are controlled through protonation states of the GFP chromophore (p-hydroxybenzylideneimidazolinone). However, of three protonation sites in the chromophore, only two have been studied. To understand the structural origin of the observed spectrophotometric and fluorescence characteristics of GFPs, employing ab initio methods, we have investigated all the possible protonation sites of the chromophore of denatured GFPs under different pH conditions. Our results suggest that the denatured GFP chromophore exists in not just two protonation states, as widely assumed in the literature, bur in five different protonation states that depend on pH over the range -3.2. to 9.4 as assessed from the predicted pK(a) values and the self-consistent reaction field continuum calculations of solvation employing Schrodinger's Jaguar 3.5 program. The unexpected complexity of the protonation states of the denatured GFP chromophore postulated here may provide a useful starting point Tor a further investigation of the protonation states of the intact GFP chromophore responsible for the experimentally observed UV absorption and fluorescence emission properties of structurally intact GFPs.