A mixed culture dechlorinating 1,2-dichloroethane (1,2-DCA) to ethene was enriched from groundwater that had been subjected to long-term contamination. In the metagenome of the enrichment, a 7-kb reductive dehalogenase (RD) gene cluster sequence was detected by inverse and direct PCR. The RD gene cluster had four open reading frames (ORF) showing 99% nucleotide identity with pceB, pceC, pceT, and orf1 of Dehalobacter restrictus strain DSMZ 9455 T , a bacterium able to dechlorinate chlorinated ethenes. However, dcaA, the ORF encoding the catalytic subunit, showed only 94% nucleotide and 90% amino acid identity with pceA of strain DSMZ 9455 T . Fifty-three percent of the amino acid differences were localized in two defined regions of the predicted protein. Exposure of the culture to 1,2-DCA and lactate increased the dcaA gene copy number by 2 log units, and under these conditions the dcaA and dcaB genes were actively transcribed. A very similar RD gene cluster with 98% identity in the dcaA gene sequence was identified in Desulfitobacterium dichloroeliminans strain DCA1, the only known isolate that selectively dechlorinates 1,2-DCA but not chlorinated ethenes. The dcaA gene of strain DCA1 possesses the same amino acid motifs as the new dcaA gene. Southern hybridization using total genomic DNA of strain DCA1 with dcaA gene-specific and dcaB-and pceB-targeting probes indicated the presence of two identical or highly similar dehalogenase gene clusters. In conclusion, these data suggest that the newly described RDs are specifically adapted to 1,2-DCA dechlorination.Chlorinated alkanes are prevailing groundwater contaminants in many industrialized countries (www.epa.gov/enviro /html/tris/ez.html), and they cause serious environmental problems (14). Among these, 1,2-dichloroethane (1,2-DCA) represents one of the world's most important toxic C 2 chlorinated aquifer pollutants. From 1987 to 1993, over 209 tons of 1,2-DCA were released into groundwater (www.epa.gov/enviro /html/tris/ez.html). Enhancement of natural attenuation processes can play a major role in achieving remediation, where applicable (17, 30), and often bioremediation alone or in combination with physical treatment of the most contaminated areas could represent the most convenient solution. The main catalyzers of the bioremediation processes are microorganisms that can dehalogenate and/or mineralize the toxic molecules with their enzymatic systems (12,40).A metabolic process of particular interest is based on dehalorespiration, a reaction that couples reductive dehalogenation with energy conservation (11,35,40). Dehalorespiration is one of the key processes for the remediation of polluted groundwaters (20,24,25). The key catalysts in dehalorespiration are reductive dehalogenases (RDs), membrane-associated enzymes with low levels of nucleotide identity but with some common traits, such as two iron-sulfur clusters as prosthetic groups, a twin-arginine translocation signal peptide (TAT system), and corrinoid cofactors (22). Recently, it has been reported that ...