Optogenetics allows for spatiotemporal control of cellular processes with light. In neuroscience, microbial rhodopsins such as light-activated ion pumps and channelrhodopsins are commonly applied to alter the neuronal membrane potential, thereby tuning cellular excitability. Up to now, microbial rhodopsins have been targeted to a number of different neuronal compartments, including the postsynaptic density, dendrite and axon initial segments, thus allowing modulation of the local membrane potential. In contrast, optogenetic tools to change ion and voltage gradients along intracellular membranes have not been developed to date. We here report on a new class of optogenetic actuators that enables light-activated acidification of intracellular compartments such as synaptic vesicles and lysosomes. Moreover, these tools incorporate fluorescent pH reporters to follow light-induced pH changes in the respective organelles. For synaptic targeting we introduced the green-light activated proton pump Arch3 from Halorubrum sodomense (1) together with the red-fluorescent protein mkate2 (cytosolic side) and the pH-sensitive pHluorin (luminal side) (2) after the third helix of the vesicular marker protein synaptophysin, followed by a linker helix and the fourth synaptophysin helix. The resulting construct- in the following referred to as pHoenix- was expressed in murine hippocampal neurons using lentivirus. In neurons, pHoenix colocalizes with the vesicular glutamate transporter 2 and resides on synaptic vesicles as shown by fluorescence measurements and electrophysiology. Next, we incubated autaptic cultured neurons with the V-type ATPase inhibitor bafilomycin for 2 hours and performed whole-cell patch clamp measurements. After action potential triggering no or very small excitatory postsynaptic currents (EPSCs) were present indicating insufficient glutamate uptake in newly formed synaptic vesicles due to the lack of proton-motive force across the vesicular membrane. pHoenix activation by green light restored the proton gradient and recovered full EPSCs within 2 min. Acidification of synaptic vesicles was fast (~5s) compared to glutamate uptake (~60s) as was concluded from simultaneous pHluorin imaging and EPSC recordings. In the following pHoenix was applied to analyze vesicular release probability in dependence of the vesicular fill state. Therefore, we analyzed miniature EPSC amplitude and frequency, paired-pulse ratio and sucrose-induced vesicle release following complex illumination protocols. Our data confirms that release probability is higher for full vesicles compared to partially filled vesicles (3). Finally, we created a pHoenix variant targeted to lysosomes by replacing the synaptophysin moieties by the transmembrane domains of the lysosomal marker protein CD63 (lyso-pHoenix). Lyso-pHoenix enabled reversible lysosomal acidification with light, both in bafilomycin-treated HEK and HELA cells.