The cellular redox status contributes to cytosolic signaling by modulating various cellular proteins. Especially mitochondrial metabolism modulates the cytosolic redox status by oxidizing NADH/FADH₂ and generating reactive oxygen species (ROS). Objectives: Since redox-sensitive dyes have serios drawbacks, we evaluated the feasibility of a novel redox- sensitive fluorescent protein (roGFP1) to monitor cytosolic ROS-levels in cultured hippocampal neurons. Methods: roGFP1 responds reversibly to oxidation/reduction and it is ratiometric by excitation, thereby enabling semi-quantitative analyses. Hippocampal cell cultures and organotypic slice cultures were transiently transfected with roGFP1 expressing vectors using electroporation or lipofectamin. Results: In cultured neurons, cytosolic roGFP1 readily responded to H₂O₂, superoxide and hydroxyl-radicals. Also it reported the endogenous ROS generation during mitochondrial inhibition by either rotenone, 3 NPA, antimycin-A and cyanide or mitochondrial uncoupling by FCCP. Oxygen withdrawal decreased cellular ROS levels, followed by a secondary ROS increase upon reoxygenation. Furthermore, we verified that roGFP1 is only negligibly affected by intracellular pH or chloride-changes, is suited for multiphoton excitation microscopy, and also capable of detecting perimitochondrial ROS microdomains. Expressed in organotypic slice cultures, roGFP1 also reliably responded to oxidizing stimuli. Conclusions: In contrast to redox-sensitive dyes, roGFP enables dynamic and semi-quantitative recordings of intracellular redox status and ROS levels. This novel optical probe proved sufficiently sensitive to follow the endogeneous generation of ROS as well as subcellular ROS microdomains. Since roGFP can also be expressed in more complex preparations such as organotypic slice cultures it constitutes a valuable tool to analyze in detail the role of ROS in cellular signaling and neurodegeneration. Supported by the Deutsche Forschungsgemeinschaft (CMPB and EXC171)