Some household products have high levels of the antimicrobial 2-n-octyl-4-isothiazolin-3-one (OIT). Although the diverse effects of OIT are of concern, information regarding its cellular actions is limited. In a previous study, we found that OIT increased intracellular Zn²⁺ levels in rat thymocytes. However, because Ca²⁺ is considered the essential cation that causes cell injury and death, we examined whether Ca²⁺ and Zn²⁺ were involved in OIT-induced cytotoxicity and proposed the mechanisms underlying these results. The effects of OIT on the membrane and cellular parameters of rat thymocytes were examined with a flow cytometer and appropriate fluorescent probes. OIT (0.3-3 μM) increased intracellular Zn²⁺ levels but not intracellular Ca²⁺ levels. Therefore, the involvement of Zn²⁺ was studied further. The simultaneous application of 0.3 μM OIT and 3 μM ZnCl₂ significantly increased cells with phosphatidylserine-exposed membranes without changing the dead cells. In contrast, applications of 0.3 μM OIT or 3 μM ZnCl₂ alone had no effects. The combination of OIT (0.1-1 μM) and ZnCl₂ (1-3 μM) significantly decreased the cellular non-protein thiol contents. These changes that were induced by their combination were completely suppressed by adding an intracellular Zn²⁺ chelator. These results suggested that submicromolar concentrations of OIT induced Zn²⁺-dependent cytotoxicity in the presence of micromolar concentrations of external Zn²⁺. Because the threshold of OIT levels that affected cellular parameters in the presence of micromolar concentrations of Zn²⁺ are much lower than the OIT contents in some household products, the adverse effects of OIT are of great concern.