SUMMARY Exogenous enzyme preparations (fibrolytic activity (FIB), 0, 0.6, 1.2, 1.8, and 2.4 mg/ml liquid volume incubated; amylolytic activity (AMZ), 0, 0.05, 0.10, 0.15, and 0.20 mg/ml liquid volume incubated; proteolytic activity (PRO), 0, 0.05, 0.10, 0.15, and 0.20 mg/ml liquid volume incubated) were incubated in vitro. Their fermentation effects were assessed based on accumulated gas production, kinetic parameters, and fermentation profile using the technique of gas fermentation. Ruminal liquid was obtained from two rumen cannulated Santa Ins sheep, fed a diet with roughage-to-concentrate ratio of 20:80. Accumulated gas production was during 96 h of incubation, measured at 18 different times. After incubation, pH, dry matter degradability (DMD), organic matter in vitro digestibility (OMD), metabolisable energy (ME), partitioning factor (PF96), gas yield (GY24), short chain fatty acids (SCFA), and microbial protein production (MCP) were evaluated. Increasing FIB dose linearly decreased (P<0.05) lag time without affecting others kinetic parameters. However, FIB increased the accumulated gas production, resulting in improved DMD, OMD, ME, GY24 and SCFA. The addition of AMZ decreased linearly (P<0.05) lag time and increased (P<0.05) gas production on initial times of incubation without altering the fermentation profile. The inclusion of PRO did not affect (P>0.05) the evaluated parameters. The addition of these exogenous enzyme preparations with fibrolytic activity altered ruminal fermentation in vitro of diets containing high levels of concentrates.