To examine the role of metallothionein (MT) on heavy metal accumulation in hepatic cytosol of rats, this study was carried out to determine the relative Zn, Cd and Cu-binding capacities of MT in hepatic cytosol of Zn, Cd and Cu-injected rats, respectively. The heavy metal contents were detected in the liver and cytosol in the following proportions: 65.2-74.8% of the Zn content, 61.9-65.6% of the Cu content, and approx. 65% of the Cd content. Each rat was given a single intraperitoneal injection of saline, ZnSO4 (1, 5, 10 and 20 mg Zn/kg b.w.), CuSO4 (2, 4 and 6 mg Cu/kg b.w.) or CdCl2 (1, 2 and 3 mg Cd/kg b.w.). The amounts of the increased Zn and Cu were attributable to MT and high molecular weight proteins, while most of the increased Cd was attributable to MT. There was a close relationship between heavy metal content of the cytosol and MT in all heavy metal-injected rats. Our data demonstrated that approx. 60% of the increased Zn or Cu in the hepatic cytosol of Zn or Cu-injected rats was bound to MT, while 83% of the increased Cd in hepatic cytosol of Cd-injected rats was bound to MT. Therefore the order of relative binding capacity in vivo of MT determined for several metals (Cd > Zn > Cu) did not correlate with the published in vitro order of affinity to MT, Cu > Cd > Zn. These results suggested that the role of MT in Zn or Cu accumulation in the liver of Zn or Cu-injected rats was different from that of MT in Cd accumulation in the liver of Cd-injected rats.