c Carbon disulfide (CS 2 ) and carbonyl sulfide (COS) are important in the global sulfur cycle, and CS 2 is used as a solvent in the viscose industry. These compounds can be converted by sulfur-oxidizing bacteria, such as Acidithiobacillus thiooxidans species, to carbon dioxide (CO 2 ) and hydrogen sulfide (H 2 S), a property used in industrial biofiltration of CS 2 -polluted airstreams. We report on the mechanism of bacterial CS 2 conversion in the extremely acidophilic A. thiooxidans strains S1p and G8. The bacterial CS 2 hydrolases were highly abundant. They were purified and found to be homologous to the only other described (archaeal) CS 2 hydrolase from Acidianus strain A1-3, which forms a catenane of two interlocked rings. The enzymes cluster in a group of -carbonic anhydrase (-CA) homologues that may comprise a subclass of CS 2 hydrolases within the -CA family. Unlike CAs, the CS 2 hydrolases did not hydrate CO 2 but converted CS 2 and COS with H 2 O to H 2 S and CO 2 . The CS 2 hydrolases of A. thiooxidans strains G8, 2Bp, Sts 4-3, and BBW1, like the CS 2 hydrolase of Acidianus strain A1-3, exist as both octamers and hexadecamers in solution. The CS 2 hydrolase of A. thiooxidans strain S1p forms only octamers. Structure models of the A. thiooxidans CS 2 hydrolases based on the structure of Acidianus strain A1-3 CS 2 hydrolase suggest that the A. thiooxidans strain G8 CS 2 hydrolase may also form a catenane. In the A. thiooxidans strain S1p enzyme, two insertions (positions 26 and 27 [PD] and positions 56 to 61 [TPAGGG]) and a nine-amino-acid-longer C-terminal tail may prevent catenane formation.