Different methods for the determination of pepsin activity in human gastric juice, using defined oligopeptides as substrates, were investigated. N-Acetyl-L-phenylalanyl-L-3,5-diiodotyrosine and tripeptides like benzyloxycarbonyl-L-histidyl-L-phenylalanyl-L-tryptophan ethyl ester, benzyloxycarbonyl-L-histidyl-L-phenylalanyl-L-tyrosine ethyl ester, or benzyloxycarbonyl-L-histidyl-L-4-nitrophenylalanyl-L-phenylalanine methyl ester lead to only small absorption changes in the direct measurement of pepsin activity. Suitable substrates were shown to be the hexapeptide, L-leucyl-L-seryl-L-4-nitrophenylalanyl-L-norleucyl-L-alanyl-L- leucine-methyl ester and the octapeptide, L-prolyl-L-histidyl-L-leucyl-L-seryl-L-4-nitrophenylalanyl-L-norleucyl-L -alanyl-L-leucine methyl ester. Hydrolysis of these peptides can be measured continuously, using a spectral line photometer at 313 nm. Optimal test conditions and kinetic constants for substrate cleavage by pepsin in solution and by human gastric juice were determined.