Four experiments, designed to study the role of biotin in cellulose digestion and volatile fatty acid production by rumen microorganisms, were conducted. These included trials to measure the effects of deletion of biotin from the fermentation medium, the addition of biotin antimetabolites to the medium, and the inclusion of metabolic intermediates on cellulose digestion and volatile fatty acid production.Omission of biotin from the medium markedly decreased cellulose digestion and volatile fatty acid production. Production of propionate was decreased approximately twice as much as was the production of acetate.Inclusion of the antimetabolite, desthiobiotin, at concentrations up to 0.47 mM had no effect, but 12 units of avidin per tube resulted in decreased cellulose digestion and reduced volatile fatty acid production. The latter effects were similar to those observed when biotin was omitted from the medium. The inhibitive effect of avidin was partially reversed by adding extra biotin. This suggests that the specific effect of a biotin deficiency on volatile fatty acid production probably involves a metabolic pathway concerned with propionate production.The addition of metabolic intermediates (glucose, 3-phosphoglycerate, pyruvate, lactate, oxaloacetate, malate, fumarate, and succinate) to biotin-containing and biotin-deficient systems failed to demonstrate that a deficiency of biotin resulted in blockage of a pathway of intermediary metabolism at any specific point.