Batch and continuous culture techniques were used to evaluate the effect of the ionophore lysocellin on ruminal fermentation and microbial populations. In batch culture, .5 and 1 ppm (of the fluid) lysocellin markedly decreased (P < .01) the acetate:propionate ratio without affecting fiber digestion, ammonia concentration, or culture pH. Greater concentrations of lysocellin had negative effects (P < .05) on fiber digestion and increased (P < .05) culture pH. In continuous culture, a low level of lysocellin (33 ppm of the diet DM or about .7 ppm of the fluid) decreased pH (P < .05) and methane (P < .05) production but had no effect on fiber digestion. Lysocellin tended to increase (P < .05) OM digestion in corn-based diets but decreased OM digestion in barley-based diets (starch source x lysocellin interaction, P < .05). In addition, the molar proportion of propionate was increased more in barley- than in cornbased diets. Total anaerobes and amylolytic and lactate-utilizing microorganisms were not affected by the ionophore. In continuous culture, cellulolytic and lactate-producing organisms were insensitive to lysocellin, but, in lysocellin-treated media, cellulolytic organisms were resistant, whereas lactic acid producers were sensitive to lysocellin at 4 ppm. In summary, the ionophore lysocellin alters ruminal fermentation by decreasing ruminal methane production and increasing the molar proportion of propionate; however, effects varied depending on whether corn or barley served as the primary starch source.