A metabolism trial was conducted to study the effect of two direct-fed microbial cultures (Saccharomyces cerevisiae, SC; Aspergillus oryzae, AO) and neutral detergent fibre (NDF) level on ruminal fermentation, Six ruminally fistulated Holstein heifers (300 kg body weight) were randomly assigned to a 6 X 6 Latin square where treatments were control groups (CG) at two NDF levels (27 and 37%), and combinations with S. cerevisiae (0 or 10 g) or A. oryzae (0 or 3 g) cultures. Animals were restricted fed 10.5 kg dry matter day(-1) (08:00 and 20:00 h). Diets were based on alfalfa hay (40 or 60%), barley grain (42.1 or 20.3%) and corn stover (1.4 or 9.4%). NDF levels of 27% and 37% gave rise to significantly different ruminal pH values (6.21 vs. 6.49, P < 0.01) and volatile fatty acid concentrations (73.0 vs. 66.4 mmol l(-1), P < 0.05). There were interactions (P < 0.05) between microbial cultures and NDF level at 6, 9 and 12 h for propionate molar percentage and entodiniomorphid population. In situ alfalfa NDF disappearance at 48 h incubation was increased (P < 0.05) by microbial cultures (CG, 53.8(b); SC, 56.7(a,b); AO, 59.2%(a)), but was reduced (P < 0.01) by the lower NDF level (54.4% vs. 58.7% for 27% and 37% NDF, respectively). According to these results, Saccharomyces cerevisiae and Aspergillus oryzae cultures increased in situ alfalfa NDF digestion at 48 h and propionate concentration at both levels of NDF.