We present evidence that two distinct regions of the DNA upstream from the mouse metallothionein-I gene contain metal-responsive regulatory sites. This result was obtained by analyzing a systematic series of deletion, insertion, duplication, and clustered point mutations introduced into cultured cells on a simian virus 40 plasmid vector. The two upstream regions contain a duplicated evolutionarily conserved DNA sequence. While either upstream region is sufficient to confer heavy metal responsiveness, both are required to give maximal levels of induced transcription.