Metallothioneins are small, cysteine-rich proteins that avidly bind heavy metals such as zinc, copper, and cadmium to reduce their concentration to a physiological or nontoxic level. Metallothionein gene transcription is induced by several stimuli, notably heavy metal load and oxidative stress. Transcriptional induction of metallothionein genes is mediated by the metal-responsive transcription factor 1 (MTF-1), an essential zinc finger protein that binds to specific DNA motifs termed metal-response elements. In cell-free DNA binding reactions with nuclear extracts, MTF-1 requires elevated zinc concentrations for efficient DNA binding but paradoxically is inactivated by other in vivo inducers such as cadmium, copper, and hydrogen peroxide. Here we have developed a cell-free, MTF-1-dependent transcription system which accurately reproduces the activation of metallothionein gene promoters not only by zinc but also by these other inducers. We found that while transcriptional induction by zinc can be achieved by elevated zinc concentration alone, induction by cadmium, copper, or H 2 O 2 additionally requires the presence of zinc-saturated metallothionein. This is explained by the preferential binding of cadmium or copper to metallothionein or its oxidation by H 2 O 2 ; the concomitant release of zinc in turn leads to the activation of transcription factor MTF-1. Conversely, thionein, the metal-free form of metallothionein, inhibits activation of MTF-1. The release of zinc from cellular components, including metallothioneins, and the sequestration of zinc by newly produced apometallothionein might be a basic mechanism to regulate MTF-1 activity upon cellular stress.All living organisms are able to cope with a variety of stress situations by immediately adapting their gene expression program to the stress stimulus. For example, metallothioneins, small cysteine-rich proteins, are strongly upregulated upon heavy metal load (2,27,29,49,63,70,71). They have the ability to bind and hence neutralize toxic (such as Cd and Hg) and excess nontoxic (such as Zn and Cu) heavy metal ions and also act as radical scavengers. Metal response element binding transcription factor 1, also called metal-responsive transcription factor 1 (MTF-1), plays an important role in the cellular response to heavy metal stress (2,21,38,55,75) and is essential for embryonic liver development in the mouse (24). MTF-1 contains six zinc fingers of the C 2 H 2 type. C-terminal to the zinc fingers are three distinct activation domains, an acidic, a proline-rich, and a serine/threonine-rich domain. Via the zinc fingers, it binds to DNA sequence motifs with the consensus binding site TGCRCNC, known as metal response elements (MREs). MREs are present in the promoters of metallothionein genes (41,61,64) and other target genes, including zinc transporter ZnT1 and â„-glutamylcysteine synthetase heavy chain (24,34,37). MTF-1 is also involved in the responses to oxidative stress (16, 24), hypoxia (23, 46), and amino acid starvation (1). In quiescent cells, MTF-1 pref...