This study examined the ability of different electron donors (i.e., hydrogen, methanol, butyrate, and yeast extract) to sustain long-term (500 days) reductive dechlorination of tetrachloroethene (PCE) in anerobic fill-and-draw bioreactors operated at 3:1 donor:PCE ratio (defined on a total-oxidation basis for the donor). Initially (i.e., until approximately day 80), the H2-fed bioreactor showed the best ability to completely dechlorinate the dosed PCE (0.5 mmol/L) to ethene whereas, in the presence of methanol, butyric acid or no electron donor added (but low-level yeast extract), dechlorination was limited by the fermentation of the organic substrates and in turn by H2 availability. As the study progressed, the H2-fed reactor experienced a diminishing ability to dechlorinate, while more stable dechlorinating activity was maintained in the reactors that were fed organic donors. The initial diminished ability of the H2-fed reactor to dechlorinate (after about 100 days), could be partially explained in terms of increased competition for H2 between dechlorinators and methanogens, whereas other factors such as growth-factor limitation and/or accumulation of toxic and/or inhibitory metabolites were shown to play a role for longer incubation periods (over 500 days). In spite of decreasing activity with time, the H2-fed reactor proved to be the most effective in PCE dechlorination: after about 500 days, more than 65% of the added PCE was dechlorinated to ethene in the H2-fed reactor, versus 36%, 22%, and <1% in the methanol-fed, butyrate-fed, and control reactors, respectively. © 2005 Wiley Periodicals, Inc.