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 Holstem 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 6.71-6.76 (p<0.0001), a linear decrease in ammonia concentrati on from 15.84-11.77 mmolL-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 41.14-33.54 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. © Medwell Journals, 2010.