The CUP1 locus in yeast confers resistance to copper toxicity. We determined the molecular basis for copper resistance in three yeast strains, with differing degrees of resistance. Increased resistance to copper is associated with overproduction of a low molecular weight copper-binding protein, copper-chelatin. Increased chelatin synthesis results from amplification of the CUP1(r) gene and increased synthesis of the copper inducible mRNA. The copper resistance level of a given strain correlates directly with the gene copy number.Strains containing one copy and ten tandemly iterated copies of the CUP1 gene were studied. From the latter, a haploid strain with enhanced resistance was isolated following several selection cycles at elevated copper concentrations. This strain was disomic for chromosome VIII, the chromosome containing the CUP1 locus. The disomic chromosomes exhibit differential CUP1 gene amplification: 11 and 14 tandemly organized repeat units are found in the respective chromosome VIII homologues. We propose that the molecular mechanisms of gene amplification involve unequal sister chromatid exchange and intrachromosomal gene conversion, as well as disomy.