Aspergillus oryzae fermentation extract (AO; 2 g/day) was added to the diet of sheep fed grass hay supplemented with 30 or 70% barley. AO decreased the proportion of propionate in rumen volatile fatty acids with both levels of supplementation (146 and 163 mmol/mol with AO v. 157 and 186 mmol/mol with no addition for the low and high barley diets respectively; P < 0·05), and also caused a small reduction in L-lactate concentration in the high barley diet (1·15 mM v. 1·43 mM in the absence of AO). Rumen pH was not changed significantly. Total viable bacteria in the rumen were stimulated with AO (2·12 and 2·46 v. 0·97 and 1·80 × 109/ml respectively). Numbers of cellulolytic bacteria and ciliate protozoa were unchanged. Hay suspended in nylon bags in the rumen tended to be degraded more rapidly with AO, but the effect was not statistically significant. Neither barley nor AO significantly altered the plasma concentrations of glucose, urea, insulin, gastrin or cholecystokinin. It was concluded that the effects of AO were generally similar for both levels of barley supplementation and that changing the composition of the diet had no detectable effect on the measured indicators of nutritional status in blood.