The Escondida mine, located in northern Chile, is the largest copper producing mine in the world. It has an abundant low-grade (ca. 0.5% Cu) sulfide copper ore reservoir, which is processed in large heap bioreactors at the mine. To understand better how microorganisms adapt to heap leaching environments, we have isolated and identified acidophiles from pregnant leach solution (PLS) from the heaps. Six bacteria and one archaeon were isolated directly on solid overlay media, and identified by phylogenetic analyses of their 16S rRNA genes as strains of Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, Leptospirillum ferriphilum, Acidiphilium cryptum and Ferroplasma acidiphilum. The sequences of the 16S rRNA genes from isolated strains showed high similarity with those detected previously by culture-independent analyses performed on samples from a pilot plant for this process. Of the three known species of Leptospirillum, only L. ferriphilum has been detected in Escondida PLS. Tolerance of the Escondida isolate (coded IESL-25) to copper and some other transition metals such as zinc, nickel and silver was compared with several other strains of both L. ferriphilum and Leptospirillum ferrooxidans. It was noted that all L. ferriphilum strains (including IESL-25) displayed far greater tolerance to both copper and silver than strains of L. ferrooxidans, though tolerance to zinc and nickel was similar among isolates of both species. Micro-representational-difference analysis (MRDA) was used to study the genetic response of L. ferriphilum IESL-25 to high copper concentration. Gene sequences obtained by MRDA were analyzed using available genomic information for L. ferriphilum and one copper-induced gene identified appears to be involved in lipopolysaccharide biosynthesis.