Novel varieties of perennial ryegrass with high water soluble carbohydrate (WSC) concentrations have been bred to increase energy availability in the rumen. In red clover (RC) the activity of polyphenol oxidase (PPO) can help protect protein in the rumen by decreasing or delaying proteolysis. Both factors (WSC and PPO) offer potential to improve the synchronization or balance between energy and nitrogen availability for rumen microorganisms and consequently to optimise rumen microbial synthesis. However, it remains unclear if these effects can be attrib uted to differences in diet composition or to changes in the rumen microbial ecosystem. The objective of this in vitro experiment was to study how the rumen microbiota was affected by diet WSC content and PPO activity.