Effects of providing diets containing grass silage (12 g of DM/day) and barley grain (3 g DM/day) to an artificial rumen (RUSITEC) in which the grass was not inoculated (GS, control), or inoculated with Lactobacillus plantarum CCM 4000 (GS + LP), Lactobacillus fermentum LF2 (GS + LF) or Enterococcus faecium CCM 4231 (GS + EF) on rumen metabolism was examined. Diets were fermented in fermentation vessels for 12 days, which included a stabilization period of 6 days. Although treatment GS + EF reached the highest cell counts among the 3 silage inoculants evaluated, GS + LP had the greatest alterations in rumen fermentation patterns and lipid metabolism. Dry matter and organic matter degradabilities were increased (P<0.05) during fermentation of the GS + LP diet, and production of propionate (P<0.001) also increased. The decrease in total production of volatile fatty acids (VFA, P<0.001, P<0.01) occurred in the grass silages inoculated with the strains CCM 4000 and LF2 (GS + EF, GS + LF) in comparison to the GS diet. Production of acetate (P<0.001, P<0.01) and n-butyrate (P<0.001) was also decreased. However, the inoculated grass silages diets did not have altered methane production or efficiency of microbial protein synthesis in RUSITEC. Fermentation of GS + LP diet decreased biohydrogenation of C18:2 and C18:3 (P<0.001) in the rumen fluid and increased (P<0.001) the concentration of cis 9, trans 11 C18:2 (CLA). In contrast, the grass silage diets inoculated with the inoculants L fermentum LF2 and E. faecium CCM 4231 (GS + LF, GS + EF) increased biohydrogenation of C18:2 (P<0.001) and C18:3 (P<0.05, P<0.001) in rumen fluid. Concentrations of CIA and TVA decreased (P<0.001) during fermentation of the GS + EF and GS + LF diets in comparison to the GS diet. Only L plantarum CCM 4000 increased CIA concentration (about 0.82 mg/day) in fermentation fluid in RUSITEC. (C) 2008 Elsevier B.V. All rights reserved.