Leu-enkephalin (YGGFL) and several analogues were chosen as model peptides for the study of peptide absorption and hydrolysis in the rat jejunum. An HPLC assay was adapted to detect YGGFL or the analogues and metabolites. Peptide hydrolysis was studied in the rat jejunum using a single-pass perfusion method. Extensive hydrolysis of YGGFL was observed in the rat jejunum and approaches to reduce its metabolism were studied. The brush border enzymes are a major site of enkephalin hydrolysis. Lumenal peptidases were secondary to the brush border enzymes in hydrolyzing the enkephalins in this system. In the in situ perfusion system, YGGFL is hydrolyzed primarily to Tyr and GGFL by the brush border aminopeptidase and to YGG and FL by brush border endopeptidase. Lowering the jejunal pH below 5.0 significantly reduces aminopeptidase activity and, to a lesser extent, endopeptidase activity. An aminopeptidase inhibitor, amastatin, produced more pronounced inhibitory effects at higher pH and the endopeptidase inhibitors, tripeptides YGG and GGF, are effective even below pH 5.0. Coperfusion of YGGFL with a combination of aminopeptidase and endopeptidase inhibitors, e.g., amastatin and YGG, is more effective in inhibiting hydrolysis since both metabolic pathways are inhibited. Leu-D(Ala)2-enkephalin, while showing enhanced stability against aminopeptidase hydrolysis, is hydrolyzed at the Gly-Phe bond by the endopeptidase. Its hydrolysis is not affected by pH changes or amastatin but is decreased by YGG. The YGGFL wall permeability was estimated and is not a limiting factor for oral absorption.