Proteolytic strains of Bacteroides amylophilus, Bacteroides ruminicola, Butyrivibrio fibrisolvens, Butyrivibrio alactacidigens, Selenomonas ruminantium var. ruminantium, Se. ruminantium var. lactilytica and of the genera Eubacterium, Fusobacterium and Clostridium were isolated from the rumen of sheep. The location of the proteolytic enzymes, their activity against various substrates and their sensitivity to inhibitors were compared with the same properties of mixed bacteria prepared from stained rumen fluid, in order to assess the relative importance of the different species in the hydrolysis of protein in the rumen. Bacteroides ruminicola was judged by these criteria to be the most important of these proteolytic isolates in vivo. Other isolates of higher specific activity had enzymes with properties different from rumen fluid. Species of Butyrivibrio, Selenomonas and Clostridium of lower specific activity may also be important if present in sufficiently large numbers, as their activity was of the same type as the mixed population. Streptococcus bovis had a low proteolytic activity as determined by the conversion of C-labelled casein to TCA-soluble products, but was able to grow on casein, probably by virtue of its exceptionally high leucine aminopeptidase activity.