Metallothioneins (MTs) are low molecular weight metal binding proteins that are implicated in heavy metal detoxification processes, and their quantification are commonly used as biomarkers of exposure to heavy metal ions. To verify the use of zebrafish MT gene promoter for aquatic toxicology studies, an 835 bp zebrafish MT (zMT) gene promoter with four putative metal regulatory elements (MREs) was isolated by DNA walking method and characterized for metal induction of gene transcription using transient expression assays in a zebrafish caudal fin cell line, SJD.1. The cloned promoter was linked upstream to a luciferase reporter gene and found to be responsive to administrations of Zn2+, Cd2+, and Cu2+ in SID.1 cell-line, but Hg2+, Ni2+, Pb2+, and Co2+ showed no significant induction of zMT gene transcription.