TsrE, a 32kDa sulfur-transferase with two rhodanese domains, was identified as a result of its over-expression during anaerobic tetrachloroethene respiration by the obligate gram-positive anaerobe Desulfitobacterium hafniense. The tsrE gene is part of an operon containing the classical ABCD gene arrangement belonging to the DMSO reductase family, and showing homology with the thiosulfate reductase operon (tsr). Detailed analysis of TsrE sequence features revealed the presence of a N-terminal lipo-box suggesting the insertion of the sulfur-transferase in the cytoplasmic membrane as a lipoprotein. Evidence was obtained for the attachment of TsrE to the membrane where it faces the outside of the cell. Each rhodanese domain of TsrE contains an active-site cysteine with motifs CKSGGR and CAVGAR. Heterologous expression and purification allowed the characterization of TsrE and variants, revealing that despite few differences, both rhodanese motifs are active in sulfur transfer. The influence on TsrE level of various sulfur compounds present in the medium was investigated, indicating a positive effect of thiosulfate, sulfide, sulfite and cysteine, but not sulfate. TsrE was expressed at the highest level, when D. hafniense was growing on thiosulfate as electron acceptor, suggesting that all tsr genes are co-expressed. A speculative function for TsrE is the mobilization of sulfur, although a clear role for TsrE in anaerobic tetrachloroethene respiration remains to be elucidated.