The trypsin inhibitor from Gleditsia triacanthos (L.) seeds was purified by affinity chromatography on a column with trypsin-Sepharose 4B. The isolated inhibitor is a single-chain protein with molecular weight of about 20 000. The inhibitor suppresses bovine trypsin at a molar rate of 1 : 1, but weakly inhibits chymotrypsin in a non-stoichiometric manner. Some properties of the isolated inhibitor closely resembled those of soybean trypsin inhibitor (Kunitz).