Twenty-nine strains of 14 species of rumen bacteria were screened for their ability to hydrolyse Ma,, Alas, GlyArg-4-methoxy-2-naphthylamide (GlyArg-MNA) and Leu-MNA. Several species, notably Megasphaera elsdenii, were active against Ala, , and a smaller number, including Bacteroides ruminicola, Butyrivibrio jibrisolvens, Ruminococcus pavefaciens, Lachnospira multiFa and Ruminobacter amylophilus, broke down Alas. Streptococcus bovis had an exceptionally high leucine arylamidase activity. However, only Ba ruminicola hydrolysed GlyArg-MNA. Further investigation revealed that only Ba rumnicola and Bu.fibrisolvens hydrolysed Alas to Ala, and Ala,, with little Ala, being produced, in a manner similar to rumen fluid. The activity of Bu. ruminicolu against synthetic peptidase substrates, including GlyArg-MNA, LysAla-MNA, ArgArg-MNA, GlyPro-MNA, LeuVal-MNA, and Ala,-p-nitroanilide, was similar to that of rumen fluid, whereas the activity of Bu.fibrisolvens was quite different. Since the main mechanism by which peptides are broken down in the rumen is similar to dipeptidyl aminopeptidase type I, for which GlyArgMNA is a diagnostic substrate, it was concluded that Ba ruminicola was the most important single species in peptide breakdown in the rumen.