The effect of rumen protozoa I. intestinalis and M. medium on the fermentation of lucerne hay and maize grain substrates was evaluated in vitro using rumen fluid from monofaunated wethers. I. intestinalis had a positive effect on the extent of feed degradation, especially in maize-containing substrates, but this effect was offset by an increase in methane production as compared to fauna-free or mixed-fauna fermentations. M. medium had no major influence on substrate degradation or production of fermentation products. The fermentation ability of both species, however, was differently influenced by the presence of fauna-free rumen fluid originated from defaunated or faunated animals.