Understanding the interactions between hydrogen producers and consumers in the rumen ecosystem is important for ruminant production and methane mitigation. The present study explored the relationships between rumen protozoa, methanogens and fermentation characteristics. A total of six donor sheep harbouring (F, faunated) or not (D, defaunated) protozoa in their rumens (D animals were kept without protozoa for a period of a few months (D - ) or for more than 2 years (D+)) were used in in vitro and in vivo experiments. In vitro the absence of protozoa decreased NH3 and butyrate production and had no effect on methane. In contrast, the liquid-associated bacterial and methanogens fraction of D+ inocula produced more methane than D -  and F inoculum (P < 0·05). In vivo fermentation parameters of donor animals showed the same trend on NH3 and butyrate and showed that D+ animals were high methane emitters, while D -  were the lowest ( - 35 %). The concentration of dissolved dihydrogen measured after feeding followed the opposite trend. Methane emissions did not correlate with the relative abundance of methanogens in the rumen measured by quantitative PCR, but there was a trend for higher methanogens concentration in the solid-associated population of D+ animals compared with D -  animals. In contrast, PCR-denaturing gradient gel electrophoresis profiles of methanogens' methyl coenzyme-M reductase A gene showed a clear clustering in liquid-associated fractions for all three groups of donors but fewer differences in solid-associated fractions. These results show that the absence of protozoa may affect differently the methanogen community and methane emissions in wethers.