Up to 43% of the viable bacteria from the rumen of cows fed grass and concentrates grew on a medium containing casein as the main substrate. Proteolytic counts for a cow fed on straw and concentrates or for a hay-fed cow were lower than counts for cows fed grass and concentrates, both in absolute terms and in relation to the total anaerobic count. In crude enzyme preparations derived from the rumen protozoa, amino acid arylamidase (leucine aminopeptidase)-like activity was the main proteolytic activity observed. In enzyme preparations extracted from the rumen bacteria in the presence of Triton X-100, trypsin-like activity was predominant. Amino acid arylamidase- and metal-chelating proteinase-like activity together with lower activities of carboxypeptidase A and B and a very low chymotrypsin-like activity were found as well. Studies with enzyme inhibitors showed that the bacterial trypsin-like activity was largely of the cysteine-protease type in a hay-fed cow, but in addition comprised serine-protease activity in a cow fed grass and concentrates. Total proteolytic activity of the enzymes in the bacterial fraction and the spectrum of proteolytic enzymes were found to vary with the ration.