Eight nucleotide sequences containing a single rhodanese domain were found in the Acidithiobacillus ferrooxidans ATCC 23270 genome: p11, p14, p14.3, p15, p16, p16.2, p21, and p28. Amino acids sequence comparisons allowed us to identify the potentially catalytic Cys residues and other highly conserved rhodanese family features in all eight proteins. The genomic contexts of some of the rhodanese-like genes and the determination of their expression at the mRNA level by using macroarrays suggested their implication in sulfur oxidation and metabolism, formation of Fe-S clusters or detoxification mechanisms. Several of the putative rhodanese genes were successfully isolated, cloned and overexpressed in E. coli and their thiosulfate:cyanide sulfurtransferase (TST) and 3-mercaptopyruvate/cyanide sulfurtransferase (MST) activities were determined. Based on their sulfurtransferase activities and on structural comparisons of catalytic sites and electrostatic potentials between homology- modeled A. ferrooxidans rhodaneses and the reported crystal structures of E. coli GlpE (TST) and SseA (MST) proteins, two of the rhodanese-like proteins (P15 and P16.2) could clearly be defined as TSTs, and P14 and P16 could possibly correspond to MSTs. Nevertheless, several of the eight A. ferrooxidans rhodanese-like proteins may have some different functional activities yet to be discovered.