Though nano-CuO has been classified as toxic toward aquatic microorganisms and its use in variousapplications is expected to increase in near future, its ecotoxicity is currently poorly understood. Theaim of this study was to investigate the mechanisms of nano-CuO toxicity based on the paradigm ofoxidative stress, the dynamics of response over 24 h, and the modulating effect of exposure conditionson toxicity and responses. To this end, the model microalga Chlamydomonas reinhardtii was exposed to10 mg L−1nano-CuO in five different exposure media, including two different growth media, two Good'sbuffers, and natural lake water. The measured endpoints included cell growth, morphological aspect,chlorophyll autofluorescence, oxidative stress, and membrane permeabilization. The results suggest thatagglomerated nano-CuO is toxic and that exposure media are decisive in whether or not particles or freeCu ions are the main mediators of toxicity. A significant particle effect was only observed in the Good'sbuffer 3-(N-morpholino) propanesulfonic acid. However, nano-CuO particles especially influenced thedynamics of response early in exposure, between 0 h and 5 h, suggesting that an adaptation to particlestress may occur or that particles modify the bioavailability of the free Cu ions in early exposure.