We report about two-photon activation of a photoactivatable derivative of the Aequorea Victoria green fluorescent protein (pa-GFP). This special form of the molecule increases its fluorescence intensity when excited by 488 nm after irradiation with high intensity light at 413 nm. Two-photon photoactivation produces an effective real three-dimensional (3D) localization of the molecular switching of pa-GFP in the bright state.