BHK cells were stably transformed with plasmid constructs that allowed constitutive expression of either mouse metallothionein-I (MT-I) or MT-III to determine whether these isoforms have different physiological properties. Cells expressing equivalent amounts of MT-I or MT-III could grow in 60-fold more cadmium than nontransfected cells and they were 2- to 3-fold more resistant to zinc, copper, and cobalt. The results suggest that the two MT isoforms detoxify these metals similarly. MT-III reduced the amount of zinc available to activate a zinc-sensitive reporter gene; MT-I actually increased the expression of the reporter gene at low concentrations of zinc. Cells expressing MT-I or MT-III also responded differently to zinc deficiency. When cells expressing MT-I were deprived of zinc, the amount of MT-I protein declined to undetectable levels, even though MT-I mRNA was still abundant, and cell proliferation was unaffected. In contrast, when cells expressing the MT-III gene were deprived of zinc, cell proliferation was arrested and MT-III protein persisted. These results suggest that MT-I does not compete with essential zinc-requiring proteins and is degraded, whereas MT-III competes for zinc and exacerbates zinc deficiency.