Optogenetics is a fast-evolving technique that employs photoreceptors to manipulate cellular activity with light. Most prominently, microbial rhodopsins are expressed in neuronal cells allowing for light-induced regulation of their respective membrane potential. While channelrhodopsin activation induces membrane depolarization, thereby triggering action potential firing, the hyperpolarizing effect of light-activated proton and chloride pumps is used for neuronal silencing. In order to broaden the palette of optogenetic applications, we aim for development of subcellularly targeted optogenetic actuators. These might be applied to study intracellular processes that underly information processing within and between neurons.Here, we describe the design and characterization of “pHoenix”, a fusion protein targeted to presynaptic vesicles. Following light application, pHoenix specifically triggers vesicular neurotransmitter uptake. Hence, expression of pHoenix in cultured neurons together with classical electrophysiology allows for correlation between presynaptic vesicular contents and respective postsynaptic responses. Moreover, pHoenix activation in combination with vesicular pH imaging might be used to study the kinetics of the underlying biochemical processes. Our results show how targeting of optogenetic actuators is crucial to decipher compartment-specific, cellular machineries that cannot be analyzed by global activation/inhibition of neurons.