Aminopeptidase activities of mixed faecal suspensions from four human donors and 12 of the most numerous species of human colonic bacteria were measured using alanine oligopeptides and various dipeptidyl- and amino acyl-arylamidase substrates. The pattern of hydrolysis of Ala4and Ala5in faecal suspensions, whereby Ala2was the first breakdown product, suggested that the main mechanism of peptide hydrolysis was dipeptidyl peptidase. Dipeptidylp-nitroanilides and 4-methoxynaphthylamides were broken down more rapidly than amino acyl derivatives in three out of four individuals tested, consistent with this conclusion. The predominantBacteroidesspp. of the intestine also had greater dipeptidyl peptidase activity than amino acyl aminopeptidase activity, whileBifidobacterium, Clostridium, EnterococcusandPropionibacteriumspp. had a more variable pattern of peptidase activities. Thus, peptide hydrolysis in the human intestine, as in the rumen, appears to be mainly a two-stage process which is initiated by dipeptidyl peptidases present in the most numerousBacteriodesspp.