The iron-oxidizing acidithiobacilli cluster into at least four groups, three of which (Acidithiobacillus ferrooxidans, Acidithiobacillus ferridurans and Acidithiobacillus ferrivorans) have been designated as separate species. While these have many physiological traits in common, they differ in some phenotypic characteristics including motility, and pH and temperature minima. In contrast to At. ferrooxidans and At. ferridurans, all At. ferrivorans strains analysed to date possess the iro gene (encoding an iron oxidase) and, with the exception of strain CF27, the rusB gene encoding an iso-rusticyanin whose exact function is uncertain. Strain CF27 differs from other acidithiobacilli by its marked propensity to form macroscopic biofilms in liquid media. To identify the genetic determinants responsible for the oxidation of ferrous iron and sulfur and for the formation of extracellular polymeric substances, the genome of At. ferrivorans CF27 strain was sequenced and comparative genomic studies carried out with other Acidithiobacillus spp.. Genetic disparities were detected that indicate possible differences in ferrous iron and reduced inorganic sulfur compounds oxidation pathways among iron-oxidizing acidithiobacilli. In addition, strain CF27 is the only sequenced Acidithiobacillus spp. to possess genes involved in the biosynthesis of fucose, a sugar known to confer high thickening and flocculating properties to extracellular polymeric substances.