Tolerance to Cd-induced toxicity following pretreatment with Cd is well documented, yet the exact mechanism of tolerance and role of metallothionein (MT) remains equivocal. In this study it was determined if the rapid induction of MT following injection of a challenge dose of Cd or the concentration of presynthesized MT induced by Cd pretreatment is more important for development of tolerance by ascertaining (1) whether the rates of synthesis of MT differ following injection of the challenge dose of Cd in control and Cd-pretreated rats and (2) if a relationship exists between presynthesized hepatic or renal MT and tolerance to Cd-induced lethality. Rates of hepatic and renal MT synthesis did not differ following injection of a challenge dose of Cd (2.0 mg Cd/kg, iv) in control and Cd-pretreated (2.0 mg Cd/kg, sc) rats. However, the progressive increase in concentration of MT in liver after Cd pretreatment correlated with the increase in tolerance to a lethal dose of Cd (4.0 mg Cd/kg, iv), as evidenced by a decrease in mortality in pretreated rats. A similar correlation was not observed between kidney MT levels and tolerance. Therefore, it appears that presynthesized MT in liver rather than increased synthesis of MT following injection of a second dose of Cd is responsible for Cd-induced tolerance.