Reactive oxygen species (ROS), including hydrogen peroxide (H2O2), are constantly generated as by-products of normal metabolic cellular pathways and can be overproduced in response to stress. In this study, we investigated ROS production and localization of H2O2 after salt (200 mM KCl) and osmotic (iso-osmotic sorbitol concentration) stress in the unicellular green alga Micrasterias. By means of the dye H2DCFDA and confocal laser scanning microscopy, most ROS production could be detected in KCl-treated cells when compared to sorbitol-exposed cells and controls. For ultrastructural detection of H2O2, CeCl3, which reacts with H2O2 and produces cerium perhydroxide deposits, has been used. Cerium was identified by transmission electron microscopy (TEM)-coupled electron energy loss spectroscopy (EELS) in organelles of KCl- and sorbitol-treated cells and in controls. Statistical measurements of the presence of the cerium M4,5 edge were performed in mitochondria, chloroplasts, cell walls, and cytoplasmic sites of five individual cells after each treatment. The most pronounced increase in H2O2 production was found in chloroplasts of KCl- and sorbitol-treated cells. This shows that the chloroplast reveals the strongest response in H2O2 production after stress induction in Micrasterias. Significant elevation of H2O2 production also occurred in mitochondria and cytoplasm, whereas H2O2 levels remained unchanged or even slightly decreased in cell walls of treated cells. Additionally, TEM micrographs and EELS analyses provided indirect evidence for an increased H2O2 production at the plasma membrane of KCl-treated cells, indicating an involvement of the plasma membrane NADPH oxidase in H2O2 generation.