A mixed culture of bovine rumen microorganisms was incubated anaerobically, under conditions likely to support growth, with added nitrate and in the presence of several potential hydrogen donors known to be intermediates in the rumen fermentation. Reduction of nitrate via nitrite to ammonia was observed but there were marked differences in the relative rates and extent of nitrate and nitrite reduction in the presence of different hydrogen donors. The hydrogen donors were ranked in decreasing order of overall effectiveness for the complete reduction of nitrate and nitrite: formate > hydrogen > glucose [Formula: see text] lactate > succinate. Methane production in the cultures was markedly depressed in the presence of nitrate. The identification by gas chromatography of nitrous oxide in the gas phase of cultures containing nitrate and incubated with hydrogen, glucose, or lactate showed that the microbiota was capable of denitrification. The quantitative significance of denitrification as a pathway of nitrate dissimilation by rumen microorganisms, however, is probably small.