It has long been recognized that isolates of iron- and sulfur-oxidizing acidophiles referred to as “Acidithiobacillus ferrooxidans” probably include more than one species, on the basis of differences in chromosomal GC contents and 16S rRNA gene sequences. Phylogenetic heterogeneity among these isolates was confirmed by phylogenetic analysis using the sequences of the 16S-23S intergenic spacers (ITS). Two main groups have been identified: the first includes the type strain of Acidithiobacillus ferrooxidans and a second comprises a cluster of newly isolated strains that have 98.5% 16S rRNA gene sequence identity with the type strain. Given that the new group of isolates have GC contents of 56 mol% as opposed to 58.8 mol% for At. ferrooxidansT, and that they share only 37% homologous DNA, these were given the new species name Acidithiobacillus ferrivorans. Further studies showed that, while strains of At. ferrivorans have many physiological traits in common with At. ferrooxidans, they also differ in some key characteristics. These include the ability to grow at temperatures as low as 4°C (as opposed to the lower limit of between 10 and 12°C for At. ferrooxidans) and the greater sensitivity of At. ferrivorans to low pH (minimum of 1.9 for growth as opposed to 1.3 for At. ferrooxidansT). Important genotypic differences include the fact that all strains of At. ferrivorans do not contain the archetypal rusticyanin gene (rusA), rather most contain a rusA homologue (rusB). Furthermore, the high potential iron-sulfur protein-encoding gene of all At. ferrivorans strains analyzed is more similar to the iro than to the hip gene characterized in At. ferrooxidansT. These results suggest that the iron oxidation pathways are different in At. ferrivorans and At. ferrooxidans.