Metallothioneins (MTs) are cysteine-rich proteins required for heavy metal tolerance in animals and fungi. Recent results indicate that plants also possess functional metallothionein genes. Here we report the cloning and characterization of five metallothionein genes from Arabidopsis thaliana. The position of the single intron in each gene is conserved. The proteins encoded by these genes can be divided into two groups (MT1 and MT2) based on the presence or absence of a central domain separating two cysteine-rich domains. Four of the MT genes (MT1a, MT1c, MT2a and MT2b) are transcribed in Arabidopsis. Several lines of evidence suggest that the fifth gene, MT1b, is inactive. There is differential regulation of the MT gene family. MT1 mRNA is expressed highly in roots, moderately in leaves and is barely detected in inflorescences and siliques. MT2a and MT2b mRNAs are more abundant in leaves, inflorescences and in roots from mature plants, but are also detected in roots of young plants, and in siliques. MT2a mRNA is strongly induced in seedlings by CuSO4, whereas MT2b mRNA is relatively abundant in this tissue and levels increase only slightly upon exposure to copper. MT1a and MT1c are located within 2 kb of each other and have been mapped to chromosome I. MT1b and MT2b map to separate loci on chromosome V, and MT2a is located on chromosome III. The locations of these MT genes are different from that of CAD1, a gene involved in cadmium tolerance in Arabidopsis.