Metallothioneins constitute a multigene family in the yeast Candida glabrata. Two genes, designated metallothionein-I (MT-I) and one member of the metallothionein-II family (MT-II), were cloned and sequenced previously (Mehra, R. K., Garey, J. R., Butt, T. R., Gray, W. R., and Winge, D. R. (1989) J. Biol. Chem. 264, 19747-19753). Southern analysis of the genomic DNA samples from different wild-type isolates indicated that the MT-I gene was always present as a single copy but multiple (3-9) and tandemly arranged copies of one MT-II gene were present in different strains. Strains of C. glabrata highly resistant to copper salts were obtained by repeated culturing of wild-type isolates in medium containing increasing concentrations of copper sulfate. These strains showed further stable chromosomal amplification (greater than 30 copies) of the MT-II gene. The MT-I gene remained as a single copy. Amplified copies of the MT-II gene were always arranged tandemly. One of the copper-resistant strains acquired more copies of the MT-II gene by apparent duplication of the chromosome carrying this gene. The size of the amplification unit was 1.25 kilobases. The principal MT-I and -II genes of C. glabrata were shown to map to different chromosomes by electrophoretic karyotypic analysis. The length of chromosome carrying MT-II gene increased appreciably in strains exhibiting the highest amplification of this gene. Northern analysis showed increased basal levels of MT-II mRNA in strains having highly amplified MT-II locus.