The rumen simulation technique (Rusitec) was used to evaluate the effects of barley grain treated with lactic acid and heat on rumen fermentation. The tested feed mixture consisted of 40% hay, 47% barley, 11.5% soybean meal and 1.5% mineral mix (dry matter basis). Four different treatments were tested: 1) none (control group), 2) barley treated with 1% lactic acid for 24h (LA1), 3) barley treated with 5% lactic acid for 24h (LA5), and 4) barley treated with 1% lactic acid and heat at 55C for 48h (LAH). No significant differences were observed between treatments concerning concentration of NH3, CO2 and short-chain fatty acids, as well as gas volume and counts of bacteria and protozoa. However, barley grain treated with lactic acid and heat (LAH) caused a significant reduction of methane concentration compared to barley treated with 5% lactic acid (LA5).