Acidophilic bacteria of mine origin are ideal systems for studying microbial metal resistance because of their ability to grow in the presence of high concentrations of metal salts. We have previously shown that the metal-resistant transformants obtained after transformation of Escherichia coli DH5alpha with plasmid DNA preparation from Acidocella sp. strain GS19h did not contain any plasmid suggesting chromosomal integration of the plasmid(s) (Appl Environ Microbiol 1997; 63: 4523-4527). The present study provides evidence in support of this suggestion. The pulsed field gel electrophoresis (PFGE) pattern of genomic DNA of the plasmidless metal-resistant transformants differed markedly from that of the untransformed DH5alpha strain. Moreover, when the recombinant plasmids constructed by cloning plasmid DNA fragments of the Acidocella strain GS19h in the vector pBluescript II KS+ were used to transform E. coli DH5alpha strain, no plasmid DNA was detected in some of the zinc- and ampicillin-resistant (Zn(r)Amp(r)) clones. The PFGE pattern of genomic DNA of such a transformed clone also differed markedly from that of the parent strain, suggesting chromosomal integration of the recombinant plasmid(s) containing both ampicillin- and zinc-resistance determinants. This observation was further supported by hybridization of chromosomal DNA of the plasmidless ZnrAmpr E. coli DH5alpha clone with the probes made from the plasmid DNA of strain GS19h and the vector DNA. Thus, this study corroborates our previous finding and documents the phenomenon of integration of metal-resistant determinants from the Acidocella GS19h plasmid(s) into the chromosome of E. coli DH5alpha.