This in vitro experiment was conducted to examine the effects of lactulose (0, 2 or 4%) on ruminal fermentation of mixtures differing in the forage concentrate ratio (F C, 20 80,40 60 or 60 40) Ruminal fluids collected from two Holstein steers were incubated with the mixtures for 48 h Data were subjected to 2 way ANOVA Lactulose did not alter ruminal fluid pH, ammonia and lactate concentrations, individual and total Volatile Fatty Acid (VFA) concentrations and gas production There was a linear Increase in ruminal fluid pH from 671-6 76 (p<0 0001), a linear decrease in ammonia concentration from 15 84-11 77 mmol L-1 (p<0 0001), a quadratic increase in lactate concentration from 25 51-26 91 mmol L-1 (p<0 04) as the F C ratio increased from 10 80-60 40 Moreover, increasing the forage level caused linear increases in the acetate (from 53 89-55 69%, p<0 0002) and valerate (from 3 05-3 25%, p<0 003) proportions and a linear decrease in the butyrate proportion (from 19 74-17 32%, p<0 0001) and a linear decrease in cumulative gas production from 4114-33 54 mL (p<0 0001) The effects of the F C ratio and lactulose level on gas kinetics parameters were variable Lactulose addition failed to modify alterations in rumen fermentation characteristics of the mixtures varying in the F C ratio In conclusion, lactulose fermentation in ruminal fluids had minimal effects on fermentability of the mixture consisting of different F C ratios suggesting that lactulose does not modify alterations in rumen fermentation in cattle during their growth and lactation phases.