To elucidate the molecular mechanism of metallothionein (MT) gene activation in response to various inducers, we constructed a G-less mouse MT-I promoter and transcribed in HeLa nuclear extract. The MT-I gene was transcribed efficiently in this extract and initiation of transcription occurred at the correct site (+1). Transcription of the MT-I gene was stimulated three- to fivefold in the nuclear extract from the cadmium-treated cells relative to the extract from the untreated cells. The MT-I promoter was also activated three- to fourfold by recombinant USF1, a helix-loop-helix-leucine zipper DNA binding transcription factor that recognizes the major late transcription factor (MLTF) binding site on the MT-I promoter. To our knowledge, this is the first report of the activation of MT-I promoter in vitro by a toxic metal and by the transcription factor USF.