Zinc ions generate a range of poorly soluble Zn-containing nanoparticles when added to commonly used mammalian cell culture media. The formation of these nanoparticles confounds the use of soluble Zn salts as positive controls during cytotoxicity testing of other Zn-containing nanoparticles, such as ZnO. These nanoprecipitates can either be crystalline or amorphous and vary in composition depending upon the concentration of Zn(II) within the medium. The cytotoxicity and immune system response of these nanoparticles in situ are similar to those of 30 nm ZnO nanoparticles. The low residual level of truly soluble Zn species (taken as species passing through a 2 kDa membrane) in cell culture media with serum is insufficient to elicit any appreciable cytotoxicity. These observations highlight the importance of employing appropriate controls when studying ZnO nanoparticle toxicity and suggest a re-evaluation of the conclusions drawn in some previous cytotoxicity studies.