The acidophilic heterotroph, Acidiphilium symbioticum KM2, is highly resistant to several metals and harbors three plasmids of 3.8, 7.1, and 56 kb in size. The bacterium becomes extremely sensitive to metals when it is cured of its plasmids. A mini-plasmid library was constructed by ligating the plasmid DNA fragments generated by MboI partial digestion into the BamHI site of pBluescriptII KS+. The Lac(-) Amp(r) transformants of Escherichia coli DH5alpha, isolated after transformation with the library, were counter-selected on Cu2+, Cd2+, Ni2+, and Zn2+-containing plates. Only Cd2+- and Zn2+-resistant colonies were developed, and, after screening, four types of recombinant plasmids designated as pNM201 (7.2 kb), pNM206 (3.4 kb), pNM208 (4.5 kb), and pNM215 (4.9 kb) were obtained. The DNA insert in pNM206 hybridized strongly with the 3.8-kb plasmid and weakly with the 7.1-kb plasmid of Acidiphilium symbioticum KM2. The DNA insert in pNM215 hybridized only with the 7.1-kb plasmid. These results strongly suggested that resistance to cadmium and zinc in A. symbioticum KM2 is mediated by these plasmids. The smallest insert of 422 bp in pNM206 conferring metal resistance in E. coli has no sequence similarity with the reported metal-resistant genes. All the putative ORFs are significantly rich (up to 37%) in basic amino acids, mainly arginine.