Three proteinase inhibitors (CLTI-I, -II and -III) were purified from the seeds of Canavalia lineata by DEAE-Toyopearl, hydroxyapatite, and anhydrotrypsin-Sepharose column chromatographies. All the inhibitors bound to trypsin at a 1:1 molar ratio and inhibited the enzyme with dissociation constants of 3-7 x 10(-9) M. They also showed the inhibitory activities on chymotrypsin. CTLI-I and -II had an identical M(r) of 8000 and very close isoelectric points (4.57 and 4.50), and existed mainly as trimers under physiological conditions. The high content of half-cystine residues and the high stability to pH and heat have suggested that these are Bowman-Birk type inhibitors. On the other hand, CLTI-III, with an M(r) of 20,500 was classified as a Kunitz (soybean) family inhibitor on the basis of the amino acid composition as well as the homology of its N-terminal 17 residues to other Kunitz inhibitors.