Investigations of physiological and toxicological effects of metal ions are frequently based on in vitro cell culture systems, in which cells are incubated with these ions in specialized culture media, instead of their physiological environment. This allows for targeted examination on the cellular or even molecular level. However, it disregards one important aspect, the different metal ion speciation under these conditions. This study explores the role of culture conditions in investigations with zinc ions (Zn²⁺). Their concentration is buffered by several orders of magnitude by fetal calf serum. Due to the complexity of serum and its many zinc-binding components, zinc speciation in culture media cannot be completely predicted. Still, the primary effect is due to the main Zn²⁺-binding protein albumin. Buffering reduces the free Zn²⁺ concentration, thereby diminishing its biological effects, such as cytotoxicity and the impact on protein phosphorylation. This is not limited to Zn²⁺, but is also observed with Ag⁺, Cu²⁺, Pb²⁺, Cd²⁺, Hg²⁺, and Ni²⁺. Usually, the serum content of culture media, and thereby their metal buffering capacity, is only a fraction of that in the physiological cellular environment. This leads to systematic over-estimation of the effects of extracellular metal ions when standard cell culture conditions are used as model systems for assessing potential in vivo effects.