Oligopeptides of l-alanine up to ala5, were incubated in vitro in either strained rumen fluid or suspensions of mixed rumen bacteria. The disappearance of substrates and formation of products were measured for 40 min, by which time ala3, ala4 and ala5 were almost totally hydrolysed. Ala2 was more slowly hydrolysed, and accumulated in incubations with the other peptides. The pattern of formation of ala2 but not ala3 from ala4, and ala3 and ala2 but not ala4 from ala5, suggested that the peptides were being hydrolysed by a dipeptidyl peptidase mechanism. Three different sheep receiving different diets gave similar results. Neither substrates nor products appeared to be accumulated intracellularly, except for ala2, and then only if protozoa were present. Protozoa were more active than bacteria in ala2 hydrolysis, whereas bacteria had greater activities with higher homologues. Similar preferences were observed with glycine peptides, although unlike alanine peptides gly3 and gly5 were more slowly degraded than the dimer. These experiments suggest that protozoa are of importance in the accumulation and hydrolysis of dipeptides, whereas bacteria are responsible for the breakdown of larger molecules by a dipeptidyl peptidase mechanism that does not appear to involve accumulation within the cell.