This study comprises the first report of ferrous iron oxidation by psychrotolerant, acidophilic iron-oxidizing bacteria capable of growing at 5°C. Samples of mine drainage-impacted surface soils and sediments from the Norilsk mining region (Taimyr, Siberia) and Kristineberg (Skellefte district, Sweden) were inoculated into acidic ferrous sulfate media and incubated at 5°C. Iron oxidation was preceded by an approximately 3-month lag period that was reduced in subsequent cultures. Three enrichment cultures were chosen for further work and one culture designated as isolate SS3 was purified by colony isolation from a Norilsk enrichment culture for determining the kinetics of iron oxidation. The 16S rRNA based phylogeny of SS3 and two other psychrotolerant cultures, SS5 from Norilsk and SK5 from Northern Sweden, was determined. Comparative analysis of amplified 16S rRNA gene sequences showed that the psychrotolerant cultures aligned within Acidithiobacillus ferrooxidans. The rate constant of iron oxidation by growing cultures of SS3 was in the range of 0.0162–0.0104 h−1 depending on the initial pH. The oxidation kinetics followed an exponential pattern, consistent with a first order rate expression. Parallel iron oxidation by a mesophilic reference culture of Acidithiobacillus ferrooxidans was extremely slow and linear. Precipitates harvested from the 5°C culture were identified by X-ray diffraction as mixtures of schwertmannite (ideal formula Fe8O8(OH)6SO4) and jarosite (KFe3(SO4)2(OH)6). Jarosite was much more dominant in precipitates produced at 30°C. Biotechnol. Bioeng. 2007;97:1470–1478. © 2007 Wiley Periodicals, Inc.