Experiments used unadapted mixed cultures of ruminal microorganisms in batch or continuous culture fermentation to investigate the effect of a thiopeptide, A10255, on ruminal fermentation and microbial populations. After 24h of fermentation in batch culture, addition of A10255 (.5 to 20ppm of the culture fluid) to 0, 45, 60, and 75% concentrate diets had no effect on total VFA but increased molar proportion of propionate and decreased butyrate. The molar proportion of acetate was decreased by treatment only in the 0 and 75% concentrate diets. The increase in molar proportion of propionate by 20ppm of A10255 was less than the increase caused by a similar concentration of monensin. The same concentration of A10255 (20ppm) decreased ADF digestion less than 20ppm of monensin. In continuous culture, A10255 (33 mg/kg of dietary DM) did not affect total VFA concentration, culture pH, OM digestion, or ADF digestion. Ruminal bacterial populations of total anaerobes and lactate-producing, lactate-utilizing, cellulolytic and amylolytic bacteria were unaffected by treatment. However, molar proportions of acetate, butyrate, and isovalerate were decreased, and propionate was increased, by addition of A10255.