Recombinant vectors containing the mouse metallothionein-I gene (MT-I) and the Escherichia coli xanthine-guanine phosphoribosyltransferase gene (gpt) were used to transfect human hgprt- HeLa cells. Transfected MT-I genes are transcriptionally regulated by cadmium but not by glucocorticoids. S1 mapping indicates that the transcripts from transfected MT-I genes begin at the correct transcription initiation site. We also transfected mouse tk- L cells with a vector containing the mouse MT- I gene and the herpes simplex virus-I thymidine kinase gene. MT-I gene transcription is regulated by cadmium but not by glucocorticoids in this homologous system as well. Finally, we fused the MT-I gene promoter/regulatory region to the thymidine kinase structural gene. Thymidine kinase activity is regulated by cadmium when this fusion gene is transfected into mouse tk- L cells. Deletion mapping experiments indicate that the DNA sequences necessary for regulation of the MT-I gene by cadmium lie within 148 bp of its transcription start site.