Optogenetics has been, and will continue to be, a boon to mechanistic studies of cellular processes. Genetically encodable proteins that sensitize the production of reactive oxygen species (ROS) are expected to play an increasingly important role, particularly in elucidating mechanisms of temporally and spatially dependent cell signaling. However, a substantial challenge in developing such photosensitizing proteins has been to funnel the optical excitation energy into the initial selective production of only one ROS. Singlet molecular oxygen, O-2(a(1)Delta(g)), is a ROS known to have a wide range of effects on cell function. Nevertheless, mechanistic details of singlet oxygen's behavior in a cell are lacking. On the basis of the rational optimization of a LOV-derived flavoprotein, we now report the development and photophysical characterization of a protein-encased photosensitizer that efficiently and selectively produces singlet oxygen at the expense of other ROS, especially ROS that derive from photoinduced electron transfer reactions. These results set the stage for a plethora of new experiments to elucidate ROS-mediated events in cells.