To test for consequences of an exposure of brain cells to copper oxide nanoparticles (CuO-NPs), we synthesised and characterised dimercaptosuccinate-coated CuO-NPs. These particles had a diameter of around 5 nm as determined by transmission electron microscopy, while their average hydrodynamic diameter in aqueous dispersion was 136 ± 4 nm. Dispersion in cell-culture medium containing 10% fetal calf serum increased the hydrodynamic diameter to 178 ± 12 nm and shifted the zeta potential of the particles from -49 ± 7 mV (in water) to -10 ± 3 mV. Exposure of cultured primary brain astrocytes to CuO-NPs increased the cellular copper levels and compromised the cell viability in a time-, concentration- and temperature-dependent manner. Application of CuO-NPs in concentrations above 100 µM copper (6.4 µg/ml) severely compromised the viability of the cells, as demonstrated by a lowered 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction capacity, a lowered cellular lactate dehydrogenase activity and an increased membrane permeability for the fluorescent dye propidium iodide. Copper internalisation as well as cell toxicity of astrocytes exposed to CuO-NPs were similar to that observed for cells that had been incubated with copper salts. The CuO-NP-induced toxicity was accompanied by an increase in the generation of reactive oxygen species (ROS) in the cells. Both, ROS formation and cell toxicity in CuO-NP-treated astrocytes, were lowered in the presence of the cell-permeable copper chelator tetrathiomolybdate. These data demonstrate that CuO-NPs are taken up by cultured astrocytes and suggest that excess of internalised CuO-NPs cause cell toxicity by accelerating the formation of ROS.