This study evaluated the effects of Quillaja saponin (0.6 and 1.2 g/L), propynoate (4 and 8 mM), and nitrate (5 and 10 mM), alone or in combinations, on methanogenesis, fermentation, bacterial and archaeal communities, and abundances of select ruminal microbial populations. All treatment decreased methane production, but combination of all three inhibitors at high dose achieved the greatest inhibition (85%). Propynoate, alone or in combination with nitrate or saponin, decreased feed degradability and total volatile fatty acid (TVFA) concentrations. However, saponin and nitrate alone at high dose and in combination at low dose inhibited methanogenesis substantially while increasing feed degradability and TVFA concentrations. The abundances of methanogens were lowered by all inhibitors except saponin alone. Fibrobacter succinogenes and Ruminococcus flavefaciens were increased by saponin, both alone and in combination with nitrate, but inhibited by propynoate. Combination of saponin and nitrate may have practical application in mitigating methane emission from ruminants. (C) 2013 Elsevier Ltd. All rights reserved.