Six reductive acetogenic bacteria from a variety of ruminal and non-ruminal environments were investigated for their ability to prevent the accumulation of methane when added to rumen fluid incubated in vitro. Acetitomaculum ruminis, Eubacterium limosum strains ATCC 10825 and ATCC 8486, Ruminococcus productus ATCC 35244, and two acetogenic bacteria, Ser 5 and Ser 8 isolated from 20 h old lambs, were grown in media containing glucose. The bacteria retained the ability to produce acetate from H-2 when incubated in the absence of sugar, while no acetate was produced in the absence of H-2. When cultures of the acetogens were added to incubations of mixed rumen microorganisms in vitro, providing between 0.05 and 0.13 mg of acetogen protein/ml, methane production decreased by about 5% after 24 h with E. limosum ATCC 8486 and Ser 5, while the other bacteria had no effect on methane production. Increasing the concentration of E. limosum ten-fold did not cause a further decrease in methane production. When E. limosum ATCC 8486 and Ser 5 were added to cultures of mixed ruminal microoganisms in the presence of 2-bromoethanesulfonic acid, which inhibited methane formation and caused H-2 to accumulate, both bacteria caused substantial increases in acetate production and decreased H-2 formation. It was concluded that although acetogenic bacteria can utilise H-2 and CO2 to form acetate in the rumen when methanogenesis is inhibited, even large concentrations of acetogenic bacteria cannot compete for H-2 with methanogenic archaea under normal circumstances. (C) 1999 Elsevier Science B.V. All rights reserved.