Our recent electrophysiological analysis of mouse retinal pigment epithelial (RPE) cells revealed that in the presence of 10 mM external thiocyanate (SCN−), voltage steps generated large transient currents whose time-dependent decay most likely results from the accumulation or depletion of SCN− intracellularly. In the present study, we investigated the effects of more physiologically relevant concentrations of this biologically active anion. In whole cell recordings of C57BL/6J mouse RPE cells, we found that, over the range of 50 to 500 µM SCN−, the amplitude of transient currents evoked by voltage steps was proportional to the extracellular SCN− concentration. Transient currents were also produced in RPE cells when the membrane potential was held constant and the external SCN− concentration was rapidly increased by pressure-ejecting 500 µM SCN− from a second pipette. Other results indicate that the time dependence of currents produced by both approaches results from a change in driving force due to intracellular SCN− accumulation or depletion. Finally, by applying fluorescence imaging and voltage-clamping techniques to BALB/c mouse RPE cells loaded with the anion-sensitive dye MQAE, we demonstrated that in the presence of 200 or 500 µM extracellular SCN−, depolarizing voltage steps increased the cytoplasmic SCN− concentration to an elevated steady state within several seconds. Collectively, these results indicate that, in the presence of physiological concentrations of SCN− outside the RPE, the conductance and permeability of the RPE cell membranes for SCN− are sufficiently large that SCN− rapidly approaches electrochemical equilibrium within the cytoplasm when the membrane voltage or external SCN− concentration is perturbed.