We have used the membrane-permeant charged fluorescent dye, 3,3'-dipropylthiadicarbocyanine iodide (diS-C3[5]), to monitor electrical potentials across the membranes of isolated bovine disks. Calibration curves obtained from experiments where a potential was created across the disk membrane by a potassium concentration gradient and valinomycin showed an approximately linear relation between dye fluorescence and calculated membrane potential from 0 to -120 mV. Light exposure in the presence of the permeant buffer, imidazole, caused a rapid decay of the membrane potential to a new stable level. Addition of CCCP, a proton ionophore, in the dark produced the same effect as illumination. When the permeant buffer, imidazole, was replaced by the impermeant buffer, Hepes, neither light nor CCCP discharged the gradient. We interpret the changes in membrane potential measured upon illumination to be the result of a light-induced increase in the permeability of the disk membrane to protons. A permeant buffer is required to prevent the build-up of a pH gradient which would inhibit the sustained proton flow needed for an observable change in membrane potential.