Live yeast cultures and yeast hydrolyzates can be used as rumen fermentation modifiers, but their effects and mode of action are different. Whereas the effects of live yeast cultures on rumen fermentation are well documented, yeast hydrolyzates have received much less attention. The influence of a yeast hydrolyzate from Saccharomyces cerevisiae, obtained after ethanol production from sugarcane (YHS), on in vitro rumen fermentation was investigated using both batch cultures and Rusitec fermenters inoculated with ruminal fluid from sheep. Batch cultures (300 mg dry matter (DM)) with two mixed diets (AHC, 0.5:0.5 alfalfa hay:concentrate; BSC, 0.15:0.85 barley straw:concentrate) as substrate were supplemented with increasing doses of YHS (0, 3.3, 6.7, 10.0 and 13.3 ml/l) and incubated for 16.5 hat 39 degrees C. Supplementation of increasing amounts of YHS to AHC-cultures increased (P < 0.05) linearly total volatile fatty acid (VFA) production and butyrate molar proportion, and decreased (P < 0.001) acetate proportion and acetate:propionate ratio. In contrast, only subtle effects of YHS on NH3-N concentrations and molar proportions of isovalerate and caproate were observed for the BSC diet. Longer-term effects of YHS supplementation on rumen fermentation of AHC diet were investigated using four Rusitec fermenters in a cross-over experimental design with two 14-day incubation periods. Fermenters were given daily 30 g of diet DM, and in each period half of them were supplemented daily with 5 ml of YHS (10.0 ml/l) Supplementing with YHS did not affect (P > 0.05) total VFA production, lactate concentrations, DM and aNDFom disappearance or enzymatic activities (amylase, xylanase and carboxymethylcellulase). Compared with the unsupplemented fermenters, YHS treatment increased (P < 0.001) NH3-N concentrations and molar proportions of propionate and butyrate at the expense of acetate, and decreased (P < 0.001) acetate:propionate ratio. In addition, YHS supplementation tended (P < 0.07) to reduce CH4/total VFA ratio and to increase microbial growth in the liquid phase of the fermenters. The automated ribosomal intergenic spacer analysis (ARISA) of samples taken on days 3, 8 and 14 of incubation from solid and liquid content of fermenters revealed that YHS supplementation increased (P < 0.02) bacterial diversity in the liquid phase and tended to increase (P < 0.08) it in the solid phase. The results indicate that YHS at a dose of 10 ml/l may be a useful dietary additive for ruminants, because it promoted a shift in fermentation toward propionate production, reduced the CH4/total VFA ratio and increased microbial growth with a 50:50 alfalfa hay:concentrate diet.