Metallothioneins (MTs) are short, cysteine-rich proteins for heavy metal homeostasis and detoxification; they bind a variety of heavy metals and also act as radical scavengers. Transcription of mammalian MT genes is activated by heavy metal load via the metal-responsive transcription factor 1 (MTF-1), an essential zinc finger protein whose elimination in mice leads to embryonic lethality due to liver decay. Here we characterize the Drosophila homolog of vertebrate MTF-1 (dMTF-1), a 791-amino-acid protein which is most similar to its mammalian counterpart in the DNA-binding zinc finger region. Like mammalian MTF-1, dMTF-1 binds to conserved metal-responsive promoter elements (MREs) and requires zinc for DNA binding, yet some aspects of heavy metal regulation have also been subject to divergent evolution between Drosophila and mammals. dMTF-1, unlike mammalian MTF-1, is resistant to low pH (6 to 6.5). Furthermore, mammalian MT genes are activated best by zinc and cadmium, whereas in Drosophila cells, cadmium and copper are more potent inducers than zinc. The latter species difference is most likely due to aspects of heavy metal metabolism other than MTF-1, since in transfected mammalian cells, dMTF-1 responds to zinc like mammalian MTF-1. Heavy metal induction of both Drosophila MTs is abolished by double-stranded RNA interference: small amounts of cotransfected double-stranded RNA of dMTF-1 but not of unrelated control RNA inhibit the response to both the endogenous dMTF-1 and transfected dMTF-1. These data underline an important role for dMTF-1 in MT gene regulation and thus heavy metal homeostasis.