Nickel oxide (NiO) is one of the important industrial materials used in electronic substrates and for ceramic engineering. Advancements in industrial technology have enabled the manufacture of ultrafine NiO particles. On the other hand, it is well-known that nickel compounds exert toxic effects. The toxicity of nickel compounds is mainly caused by nickel ions (Ni(2+)). However, the ion release properties of ultrafine NiO particles are still unclear. In the present study, the influences of ultrafine NiO particles on cell viability were examined in vitro to obtain fundamental data for the biological effects of ultrafine green NiO and ultrafine black NiO. Ultrafine NiO particles showed higher cytotoxicities toward human keratinocyte HaCaT cells and human lung carcinoma A549 cells than fine NiO particles and also showed higher solubilities in culture medium (Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum) than fine NiO particles. In particular, the concentration of Ni(2+) released into the culture medium by ultrafine green NiO was 150-fold higher than that released by fine green NiO. The concentrations of Ni(2+) released by both types of NiO particles in an aqueous solution containing amino acids were remarkably higher than those released by NiO particles in water. Moreover, we prepared a uniform and stable dispersion of ultrafine black NiO in culture medium and examined its influence on cell viability in comparison with that of NiCl(2), a soluble nickel compound. A medium exchange after 6 h of exposure resulted in a loss of cytotoxicity in the cells exposed to NiCl(2), whereas cytotoxicity was retained in the cells exposed to NiO. Transmission electron microscope observations revealed uptake of both ultrafine and fine NiO particles into HaCaT cells. Taken together, the present results suggest that the intracellular Ni(2+) release could be an important factor that determines the cytotoxicity of NiO. Ultrafine NiO is more cytotoxic than fine NiO in vitro.