Chlorinated benzenes have been extensively used in industry and agriculture and are common groundwater pollutants. Chlorobenzenes readily migrate to anaerobic zones in sediment and groundwater, thus understanding the fate of these compounds and anaerobic remediation are important goals. In previous studies in this laboratory, sediment microcosms were developed that reductively dehalogenated dichlorobenzene (DCB) isomers to monochlorobenzene (MCB) and MCB to benzene at high rates. Enrichment cultures that dehalogenated either 1,2-DCB, 1,3-DCB, or 1,4-DCB to MCB were derived from these microcosms, and Dehalobacter spp. were identified in an enrichment using 1,2-DCB. In this study, quantitative PCR (qPCR) of 16S rRNA genes indicated Dehalobacter spp. were responsible for dehalogenation in all DCB cultures, and 16S rRNA clone library analysis indicated 1,2- and 1,4-DCB dehalogenating Dehalobacter spp. were closely related while the Dehalobacter sp. in 1,3-DCB cultures was more divergent. In the course of purification of these cultures, methanogens and acetogens were eliminated, and culture conditions for growth of the dehalogenators were optimized. Extracts from a Sedimentibacter sp. isolated from a 1,2-DCB enrichment culture were added to 10-8 dilution cultures, allowing the isolation of 1,2-DCB and 1,3-DCB dehalogenating Dehalobacter sp. strains 12DCB1 and 13DCB1, although the extracts were not required for growth in subsequent transfers. Dehalobacter sp. strain 14DCB1 grew more slowly and was characterized in a highly enriched 1,4-DCB dehalogenating culture. Dehalogenation capabilities of each strain were tested with all chlorobenzene isomers, dichlorotoluenes, and tetrachloroethene (PCE), and each strain dehalogenated different combinations of these compounds. Strain 12DCB1 dehalogenated predominantly singly flanked chlorines on aromatic compounds and PCE, and did not dehalogenate compounds with only doubly flanked chlorines or those without any flanked chlorines. Strain 13DCB1 had the widest dehalogenation range of compounds tested, utilizing singly flanked chlorines, doubly flanked chlorines, meta substituted unflanked chlorines, and PCE, while strain 14DCB1 had the narrowest substrate range, dehalogenating para substituted chlorines and slowly dehalogenating some singly flanked chlorines. MCB dehalogenation to benzene was investigated in microcosms constructed with sediment from two different contaminated areas within the Chambers Works site. Attempts to transfer dehalogenation activity to sediment-free enrichment culture were not successful, though transfers into medium containing a commercial potting mix were achieved, which reduced the reliance upon limited supplies of sediment for perpetuation of MCB dehalogenation activity. A threshold below which MCB was not dehalogenated was investigated, and results suggested MCB was not utilized at concentrations below ca. 15 [MICRO SIGN]M in microcosms despite the thermodynamic favorability of the reaction. Lastly, qPCR confirmed Dehalobacter spp. played a role in MCB dehalogenation, and different Dehalobacter spp. were detected in 16S rRNA clone libraries from the two sediment types which, together with DCB culture sequences, suggested 16S rRNA gene sequence is not a good predictor of Dehalobacter spp. dehalogenation spectra. These studies have established a role for Dehalobacter spp. in the dehalogenation of chlorobenzenes, and this genus should be considered when determining the fate of diverse halogenated organic compounds.