Summary A strictly anaerobic bacterium was isolated from tetrachloroethene (PCE)‐to‐ethene dechlorinating microcosms established with river sediment without prior exposure to chlorinated solvents. The isolation procedure included the addition of 2‐bromoethanesulfonate to select against methanogenic archaea, > 50 consecutive 1–2% (v/v) transfers to reduced mineral salts medium amended with trichloroethene (TCE), acetate, and hydrogen, the addition of ampicillin, and the dilution‐to‐extinction principle. Culture‐dependent and 16S rRNA gene‐targeted approaches suggested culture purity. Microscopic examination revealed a homogeneous culture of an organism with a distinct, disc‐shaped morphology. The isolate shared > 99% 16S rRNA gene sequence similarity with members of the Pinellas group of the Dehalococcoides cluster, and was designated Dehalococcoides sp. strain FL2. Strain FL2 could be propagated with TCE, cis‐1,2‐dichloroethene (cis‐DCE), or trans‐DCE as the electron acceptors, acetate as the carbon source, and hydrogen as the electron donor in defined, completely synthetic medium. No other growth‐supporting redox couples were identified. Trichloroethene, cis‐DCE and trans‐DCE were dechlorinated at rates of 27.5, 30.4 and 18.8 µmol l−1 day−1 respectively. Quantitative real‐time polymerase chain reaction (PCR) with a fluorescently labelled linear hybridization probe confirmed growth with these electron acceptors, and suggested that strain FL2 captures energy from both the TCE‐to‐cis‐DCE and 1,2‐DCE‐to‐VC dechlorination steps. Tetrachloroethene and vinyl chloride (VC) were slowly and cometabolically dechlorinated in the presence of a growth‐supporting chloroethene, but ethene formation was incomplete, even after prolonged incubation. At room temperature, strain FL2 grew with a doubling time of 2.4 days, and yielded 166.1 ± 10.2 mg of protein per mole of chloride released. In the presence of excess electron acceptor, strain FL2 consumed hydrogen to a concentration of 0.061 ± 0.016 nM. Dechlorination ceased following the addition of 0.5 mM sulfite, whereas sulfate (10 mM) and nitrate (5 mM) had no inhibitory effects.