In vitro inoculum enriched with particle-associated organisms was prepared using rumen contents from a cow fed a 60% forage, 40% concentrate diet. Treatment of in vitro inoculum with cetyltrimethylammonium bromide was used to release intracellular free amino acids from mixed rumen organisms. Addition of 10 mM tosylarginine methyl ester, a competitive inhibitor of trypsin, decreased degradation rate and intracellular free amino acids in incubations containing either casein or serum albumin. Extracellular peptides increased rapidly to a maximum at 60 min in casein incubations but were not different from zero in albumin incubations. Accumulation of intracellular free amino acids was maximal at 60 min in casein and albumin incubations; the concentration observed with albumin was about one-fourth that with casein. Ammonia production from intact casein was slightly greater than that from acid and enzymatically hydrolyzed casein and about 80% greater than that from albumin. Ammonia production and appearance of extracellular free amino acids lagged behind accumulation of intracellular free amino acids. Results suggest that formation and metabolism of extracellular peptides are important in controlling the rate of protein degradation by mixed rumen organisms.