Every living organism must detoxify nonessential metals and carefully control the intracellular concentration of essential metals. Metallothioneins, which are small, cysteine-rich, metal-binding proteins, play an important role in these processes. In addition, the transcription of their cognate genes is activated in response to metal exposure. The zinc finger transcription factor MTF-1 plays a central role in the metal-inducible transcriptional activation of metallothionein and other genes involved in metal homeostasis and cellular stress response. Here we report that the phosphorylation of MTF-1 plays a critical role in its activation by zinc and cadmium. Inhibitor studies indicate that multiple kinases and signal transduction cascades, including those mediated by protein kinase C, tyrosine kinase, and casein kinase II, are essential for zinc- and cadmium-inducible transcriptional activation. In addition, calcium signaling is also involved in regulating metal-activated transcription. In contrast, cAMP-dependent protein kinase may not be directly involved in the metal response. Contrary to what has been reported for other transcription factors, inhibition of transcriptional activation does not impair the binding of MTF-1 to DNA, suggesting that phosphorylation is not regulating DNA binding. Elevated phosphorylation of MTF-1 is observed under condition of protein kinase C inhibition, suggesting that specific dephosphorylation of this transcription factor contributes to its activation.