Three trypsin inhibitors were isolated from tubers of taro (Colocasia antiquorum var. nymphaifolia?) and named taro trypsin inhibitors (TTI)-I, -II, and -III. The final preparations were homogeneous by polyacrylamide gel electrophoresis and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The three inhibitors showed strong and stoichiometric inhibition against bovine trypsin and the inhibitor constants (Ki) were estimated to be of the order of 10(-9) to 10(-10) M. In contrast, they had only a weak capacity to inhibit bovine alpha-chymotrypsin and did not inhibit subtilisins (BPN' and Carlsberg), porcine pepsin, or papain. Each inhibitor appeared to be a protein with a molecular weight of 40,000 which could be dissociated into two subunits, both of which had a molecular weight of 20,000. The inhibitors formed complexes with trypsin at molar ratios of 1:2, suggesting that each subunit of these inhibitors can react with the enzyme in a 1:1 molar ratio. The three inhibitors had similar amino acid compositions and none of them contained carbohydrate or free sulfhydryl group. The antitryptic activity of all three inhibitors was suppressed by treatment with 1,2-cyclohexanedione (CHD) but not with 2,4,6-trinitrobenzenesulfonate (TNBS), thus demonstrating each of the inhibitors to contain an arginyl residue at the reactive site.