AbstractChloride regulation affects brain function in many ways, for instance, by dictating the GABAergic reversal potential, and thereby influencing neuronal excitability and spike timing. Consistent with this, there is increasing evidence implicating chloride in a range of neurological conditions. Investigations about these conditions, though, are made difficult by the limited range of tools available to manipulate chloride levels. In particular, there has been no way to actively remove chloride from neurons; we now describe an optogenetic strategy, ‘Cl-out’, to do exactly this. Cl-out achieves its effect by the cooperative action of two different component opsins: the proton pump, Archaerhodopsin and a chloride channel opsin. The removal of chloride happens when both are activated together, using Archaerhodopsin as an optical voltage clamp to provide the driving force for chloride removal through the concurrently opened, chloride channels. We further show that this novel optogenetic strategy can reverse an in vitro epileptogenic phenotype.