Divalent cadmium and mercury ions are capable in vitro of displacement of zinc from metallothionein. This process has now been studied in vivo and ex vivo, using the isolated perfused rat liver system, in order to determine if this process can occur in the intact cell. Rats with normal and elevated (via preinduction with zinc) levels of hepatic zinc thionein were studied. Cd(II) completely displaces zinc from normal levels of metallothionein and on a one-to-one basis from elevated levels of metallothionein, both in vivo and ex vivo. Hg(II) displaces zinc from metallothionein (normal or elevated) rather poorly, as compared with Cd(II), in vivo, probably due to the kidneys preference for absorbing this metal. Ex vivo Hg(II) displaces zinc from metallothionein (normal or elevated) on a one-to-one basis, with considerably more mercury being incorporated into the protein than in vivo. The results of double-label ex vivo experiments using metal and [35S]cysteine (+/- cycloheximide) were consistent with the above experiments, indicating that de novo thionein synthesis was not required for short term incorporation of cadmium and mercury into metallothionein. These data are supportive of the hypothesis that cadmium and mercury incorporation into rat hepatic metallothionein during the first few hours after exposure to these metals can occur primarily by displacement of zinc from preexisting zinc thionein by a process which does not require new protein synthesis.