The final step in the conversion of protein to amino acids by the common Gram-negative rumen bacterium, Prevotella (formerly Bacteroides) ruminicola , is the cleavage of di- and tripeptides. Dipeptidase and tripeptidase activities were predominantly cytoplasmic, and toluene treatment increased the rate of Ala2 and Ala3 hydrolysis by whole cells, suggesting that transport limited the rate of hydrolysis of extracellular di- and tripeptides. The hydrolysis of Ala2 and Ala3 by whole cells was not affected by protonophores, ionophores or dicyclohexylcarbodiimide, but Ala2 hydrolysis by EDTA-treated cells was inhibited by the Ca2+/H+ ionophore, tetronasin. Ala3 hydrolysis was not affected by protonophores or ionophores in EDTA-treated cells. The dipeptidase of strain M384 was inhibited > 99% by 1,10-phenanthroline and 39% by EDTA but not other protease inhibitors, consistent with the enzyme being a metalloprotease. Tripeptidase was insensitive to protease inhibitors, except for a 33% inhibition by EDTA. Cleavage of tripeptides occurred at the bond adjacent to the N-terminal amino acid. Distinct di-, tri- and oligopeptidase peaks were obtained by anion-exchange liquid chromatography of disrupted cells. Banding patterns on native PAGE using activity staining also indicated that P. ruminicola M384 had separate single dipeptidase and tripeptidase enzymes which hydrolysed a range of peptides. The dipeptidase of strain M384 was different from other strains of P. ruminicola: strains GA33 and B14 had activities which ran at the same Rf; strain GA33 had another band of lower activity; strain 23 had two bands different from those of the other strains. The tripeptidases ran at the same Rf for the different strains. Dipeptidase activity of all strains was inhibited by 1,10-phenanthroline on gels. Gel permeation chromatography indicated that the Mr of the dipeptidases from strains M384 and B14 were 115 000 and 114 500 respectively, and 112 500 and 121 500 for the corresponding tripeptidases. Thus the metabolism of small peptides by P. ruminicola involves separate permeases and intracellular peptidases for di- and tripeptides.