An experiment was conducted to investigate the effects of defaunation on the fermentation of two different diets consisting of hay (100%) and hay+barley (80% + +20%) in a rumen pouch (RUSITEC). The survival and composition of protozoa were investigated in an in vitro system in control vessels where no defaunation was carried out. Defaunation decreased the production of total volatile fatty acids (P < < 0.01), acetate, butyrate (P < 0.001)--Tab. II; dry matter digestibility (P < 0.05) and detergent fibre digestibility: acid detergent fibre (ADF), neutral detergent fibre (NDF), cellulose (P < 0.001), hemicellulose (P < 0.01 and P < 0.001, respectively) in our experiment (Tab. I). These parameters also decreased: production of CH4 (P < 0.001), CO2 (P < 0.01 for the barley diet), total production of gases (P < 0.001 for the barley diet), total utilization (P < 0.01 and P < 0.001, respectively) and recovery of H2 (P < 0.05 and P < 0.001, respectively), NH3-N in effluent (P < 0.001 and P < 0.01, respectively)--Tab. III; and production of ATP (P < 0.01)--Tab. IV, as a result of fermentation in our experiment. On the other hand, there was a significant increase in the following parameters: production of propionate (P < 0.001 for the barley diet)--Tab. II; microbial nitrogen/kg of organic matter fermented--OMF (P < 0.001), Tab. III; energy yield--E of volatile fatty acids (VFA), proportion of VFA energy with respect to fermented hexose energy--E1, proportion of energy in bacterial cells with respect to fermented hexose energy--E3 (P < 0.001) and the effectiveness of microbial proteosynthesis--YATP (P < 0.001), Tab. IV, as a result of defaunation. The total number of protozoa and the number of some species, especially the number of big entodinio-morphid protozoa, were markedly higher (p < 0.001) for the barley diet--Tab. V. The number of protozoa decreased rapidly within three days after the system initiation. Later on, the values were more or less steady.