Two trypsin inhibitors from the seed of Acacia elata, a legume of the subfamily Mimosoideae, were isolated by affinity chromatography on trypsin-Sepharose 4B and separated by chromatography on SP-Sephadex C-25 and Sephadex G-100. The two inhibitors, with molecular weights of about 20 000, were composed of two polypeptide chains linked by a disulfide bond. The two inhibitors had essentially the same amino acid compositions and both contained four half-cystine residues, no methionine and were rich in aspartic acid, glutamic acid, glycine and leucine. The inhibitors had isoelectric points of 6.4 and 5.9. The inhibitors stoichiometrically inhibited trypsin in the molar ratio of 1:1, alpha-chymotrypsin was inhibited also in a 1:1 molar ratio but the binding of the enzyme by the inhibitor was weaker and the inhibitor-chymotrypsin complex dissociated during the assay. Both enzymes are probably inhibited at an identical site. Amino-terminal sequence analysis of the two polypeptide chains of the inhibitors revealed extensive homology with the trypsin inhibitor from the silk tree (another Mimosoideae legume); both these inhibitors are homologous with the soybean trypsin inhibitor (Kunitz).