A rumen simulation device (Rusitec) was used to compare the effects of autoclaved and γ-irradiated Aspergillus oryzae fermentation extract (AO) with those of untreated AO on rumen fermentation. Each vessel received daily 18 g of a basal diet consisting of 500 g hay, 299–5 g rolled barley, 100 g molasses, 91 g fishmeal and 9–5 g of a minerals and vitamins mixture/kg dry matter. AO preparations (0.25 g/day) were added with the feed. Dry matter digestion increased at 24 h, but not at 48 h, after adding AO, suggesting that AO stimulated the rate of digestion but not the extent. AO increased the numbers of total and cellulolytic bacteria by 90 and 50 respectively. Irradiated AO stimulated numbers of bacteria in the same way as AO, although to a slightly lesser extent. Autoclaved AO had no effect. Thus, the mode of action of AO on rumen fermentation depends on a heat-labile component, possibly a nutrient or an enzyme, or metabolic activity. Viable AO cells are not required to stimulate bacterial growth and activity.