Three acidophilic. chemolithotrophic and ferrous oxidizing bacteria were isolated from the Agrio River in the geothermal system of Copahue volcano in Neuquen (Patagonia, Argentina) using 9K enrichment medium and then purified on solid ferrous-agarose medium. Amplified ribosomal DNA restriction enzyme analysis (PCR/ARDRA) showed that these strains can be considered as Acidithiobacillus ferrooxidans. Ferrous iron oxidation of these strains, including a collection strain was compared at different temperatures (20, 25, 30, 35 and 40 degreesC) and pH values (1.6, 1.8 and 2.3). Isolated bacteria proved to be less susceptible to low temperatures or high medium acidity. These strains showed adequate growth in iron-free 9K medium using sulphur as a sole energy source. Iron(II) oxidation inhibition in the presence of different heavy metals (Ag+, Cd2+, Cu2+, Zn2+) or different anions (NO3-, Cl-) were also investigated. (C) 2004 Elsevier Ltd. All rights reserved.