The toxicological effects of zinc oxide nanoparticles (ZnO-NPs) are attracting increasing concern as the field of nanotechnology progresses. Although the literature suggests that toxicity of ZnO-NPs may be related to their dissolution, the mechanism for ZnO-NP perturbation of cytosolic zinc concentration (Zn(2+)) homeostasis remains obscure. Using FluoZin-3 and RhodZin-3, this study investigated changes in both Zn(2+) and mitochondrial free Zn(2+) concentration (Zn(2+)) under conditions of ZnO-NP treatment in vivo and in vitro. In human leukemia Jurkat cells and human lung carcinoma H1355 cells, ZnO-NP treatment resulted in an elevation of both Zn(2+) and Zn(2+). In H1355 cells, ZnO-NP treatment induced depolarization of mitochondrial membrane potential, as well as caspase-3 activation and lactic dehydrogenase (LDH) release. In our in vivo experiments, when rats were exposed to ZnO-NPs, higher Zn(2+) and Zn(2+) were recorded in both broncho-alveolar lavage (BAL) cells and white blood cells isolated from ZnO-NP-exposed rats, compared with high efficiency particulate air-filter-protected controls LDH levels were also elevated in the BAL of ZnO-NP-exposed rats compared with controls. A mechanical toxicological pathway for ZnO-NP toxicity is suggested by these results: an elevation in Zn(2+) resulting from ZnO-NP dissolution in the intracellular endosome; cytosolic Zn(2+) sequestration by mitochondria; and elevated Zn(2+) leading to mitochondrial dysfunction, caspase activation, and cell apoptosis. We conclude that exposure to ZnO-NPs interferes with the homeostasis of Zn(2+) and that elevated Zn(2+) results in cell apoptosis.