Six obligate anaerobic bacterial isolates (195, CBDB1, BAV1, VS, FL2 and GT) with strictly organohalide-respiring metabolisms were isolated from chlorinated solvent-contaminated aquifers, contaminated and uncontaminated river sediments or anoxic digester sludge. Cells were non-motile with a disc-shaped morphology of 0.3 to 1 μm in diameter, a thickness of 0.1-0.2 μm, and characteristic indentations on opposite flat sides of the cell. Growth occurred in completely synthetic, reduced medium amended with a haloorganic electron acceptor (i.e., mostly chlorinated but also some brominated compounds), hydrogen as electron donor, acetate as carbon source, and vitamins. No other growth-supporting redox couples were identified. Aqueous hydrogen consumption threshold concentrations were <1 nM. Growth ceased when vitamin B12 was omitted from the medium. Addition of sterile cell-free supernatant of Dehalococcoides-containing enrichment cultures enhanced dechlorination and growth of strains 195 and FL2, suggesting the existence of so far unidentified stimulants. Dechlorination occurred between pH 6.5 and 8.0 and over a temperature range of 15 to 35°C, with an optimum growth temperature between 25 and 30°C. The major PLFA were 14:0 (15.7 mol%), br15:0 (6.2 mol%), 16:0 (22.7 mol%), 10Me16:0 (25.8 mol%), and 18:0 (16.6 mol%). Unusual furan fatty acids including 9-(5-pentyl-2-furyl)-nonanoate (Fu18:2ω6) and 8-(5-hexyl-2-furyl)-octanoate (Fu18:2ω6) were detected in strains FL2, BAV1 and GT, but not in strains 195 and CBDB1. The 16S rRNA gene sequences of the six isolates shared greater than 98% identity, and phylogenetic analysis revealed an affiliation with the Chloroflexi phylum and a greater than 10% sequence divergence from other described isolates. The genome sizes and G+C contents ranged from 1.34 to 1.47 Mbp and 47 to 48.9 mol% G+C, respectively. Based on 16S rRNA gene sequence comparisons, genome-wide average nucleotide identity (ANI) and phenotypic characteristics, the organohalide-respiring isolates represent a new genus, for which the name Dehalococcoides gen. nov. is proposed. Isolates BAV1 (ATCC BAA-2100 = JCM 16839 = KCTC 5957), FL2 (ATCC BAA-2098 = DSM 23585 = JCM 16840 = KCTC 5959), GT (ATCC BAA-2099 = JCM 16841 = KCTC 5958), CBDB1, 195 (ATCC BAA-2266 = KCTC 15142), and VS are considered strains of the novel species Dehalococcoides mccartyi gen. nov., sp. nov. Dehalococcoides mccartyi sp. nov. strain 195T is designated as the type species of the genus.