An anaerobic mixed bacterial culture was enriched for bacteria dechlorinating 1,2,3- and 1,2,4-trichlorobenzene (TCB) to dichlorobenzenes by exclusive use of non-fermentable substrates and the application of vancomycin. Growth and dechlorination occurred in a purely synthetic medium with formate or hydrogen, acetate, and TCB. Neither acetogenesis nor methanogenesis was detected in the culture. Repeated subculturing maintaining high dechlorinating activities was also achieved when only hydrogen and TCB were supplied. This indicated that reductive dechlorination of TCB was the primary energy conservating process. The number of dechlorinating bacteria was strictly limited by the amount of TCB supplied in the medium. In addition, the dechlorinating activity could be maintained only in the presence of TCB. A most probable number analysis showed that the dechlorinating species amounted to at least 6 x 10(5) cells per ml at a total cell number of about 2 x 10(6) cells per ml. Vitamin B12 significantly stimulated the dechlorinating activity.