Photoactivatable fluorescent proteins have become an important addition to the set of molecular probes used to understand cellular function. Known as “molecular highlighters”, their fluorescence is switched on by irradiation, thereby enabling non-invasive tracking of protein trafficking and dynamics. They are also the basis for the imaging technique called photoactivated light microscopy (PALM) in which multiple emitted photons are observed from individual active fluorophores which are sequentially activated from a large pool of inactive proteins and then photobleached. By locating the center of the point spread function for these emitted photons, it is possible to determine the location of the active fluorophore at better resolution than the theoretical diffraction limit. Previous green fluorescent protein (GFP) mutants exhibiting photomodulatory behavior have been reported including Kaede and KikGR whose emission wavelength change irreversibly, Dronpa whose fluorescence can be reversibly activated with light, and photoactivatable GFP (paGFP), whose excitation wavelength can be irreversibly changed with light. For Kaede, KikGR, and Dronpa, the precise three-dimensional structure of the fluorophore must be preserved to maintain photoswitching behavior. In paGFP, the shift in excitation wavelength is mediated by a light dependent decarboxylation of Glu222. The loss of this carboxy group is believed to cause reorientation of an internal hydrogen bond network, which changes the protonation state of the fluorophore and leads to an irreversible shift in the excitation maximum from 397 nm to 475 nm. This mechanism of GFP photoactivation requires the preservation of active-site residues His203, His148, Ser205, and Glu222. Unfortunately, the sequence restrictions necessary to maintain photomodulatory behavior are not always compatible with the mutations necessary to produce other fluorescent protein variants including YFP and RFP. In addition, Kaede, KikGR, and paGFP exist in two forms with differing excitation and emission wavelengths, precluding the simultaneous use of these wavelengths for other purposes. To address these problems, we have developed a general strategy for photoactivating GFP based upon nonnatural amino acid mutagenesis with the photocaged tyrosine analogue o-nitrobenzyl-O-tyrosine (ONBY). Replacing the fluorophore tyrosine 66 with ONBY yields a GFP molecule that is non-fluorescent as observed for other onitrobenzyl appended fluorophores including fluoresceine, Texas Red, and quantum dots. An earlier nonnatural variant of GFP was produced using a similar strategy, however, the high pre-irradiation background fluorescence and low protein yield made this unsuitable for use as a molecular marker. Fast, time-resolved UV/Vis spectroscopy measurements indicate that the fluorescence quenching likely occurs through photo-induced electron transfer (PET). Irradiation at 365 nm is sufficient to remove the o-nitrobenzyl