The purpose of this study was to determine if Zn pretreatment could protect rat primary hepatocyte cultures from the cytotoxicity of five metals that have little or no affinity for metallothionein (MT). Hepatocytes were grown in monolayer cultures for 22 h and subsequently treated with ZnCI2(100 μM) for 24 h; which increased the MT concentration 15‐fold. Following Zn pretreatment, hepatocytes were exposed to various concentrations of Mn, V, Cr, Se, or Fe for an additional 24 h. Cytotoxicity was assessed by enzyme leakage and loss of intracellular K+. The toxicity of all five metals was significantly reduced in the Zn‐pretreated cells. Zn pretreatment had no appreciable effect on the hepatocellular uptake (1–24 h) of Mn or Se. Zn pretreatment also did not increase the distribution of Mn or Se to the cytosol and neither metal was bound to MT, suggesting the protection was not due to their binding to MT. However, Zn pretreatment significantly decreased Mn‐, Cr‐, and V‐induced cellular glutathione depletion. In summary, Zn pretreatment of rat primary hepatocyte cultures protects against Cr‐, Mn‐, Fe‐, Se‐, or V‐induced hepatotoxicity. This protection does not appear to be related to MT induction but may be due to Zn‐induced thiol or membrane stabilization and/or other biological changes produced by Zn.