The identification of physicochemical factors that govern toxic effects of nanomaterials (NMs) is important for the safe design and synthesis of NMs. The release of metal cations from NMs in cell culture medium and the role of the metal cations in cytotoxicity are still under dispute. Here, we report that removal of NMs such as ZnO nanoparticles (NPs) by centrifugation, the procedure commonly used for the estimation of released ion concentration in nanotoxicology, was incomplete even at a relative centrifugal force of 150 000 × g. In this sense, the Zn concentration in supernatant measured by inductively coupled plasma-mass spectrometry cannot be regarded as the concentration of free Zn2+ ions which were released from ZnO NPs in cell culture medium. This suggests the urgent need to develop relevant analytical techniques for nanotoxicology. The toxic contribution of released Zn2+ ions to the A549 cell lines was estimated to be only about 10%. We conclude that the cytotoxicity associated with ZnO NPs is not a function of the Zn concentration, suggesting that other factors play an important role in the toxic effect of ZnO NPs.