We report for the first time the isolation and characterization of a protease inhibitor from the seeds of Pithecellobium dulce, which is a Leguminosae tree native to Mexico. The purification of the P. dulce trypsin inhibitor (PDTI) was a direct process. After its extraction (pH 8.0) and precipitation (80% (NH(4))(2)SO(4)), the pH was adjusted to 4.0, the supernatant was loaded onto a CM-Sepharose column, and a single peak of trypsin inhibitory activity was eluted (CM-TIA). The main component of CM-TIA was PDTI, a protein composed of two polypeptide chains joined by disulfide bridge(s), with a pI of 4.95 and a molecular weight determined by electrospray mass spectrometry of 19 614 Da. The N-terminal sequence of PDTI has the highest similarity with the seed inhibitor of Acacia confusa. PDTI lacks chymotrypsin inhibitory activity. A low rate of cytotoxicity of CM-TIA toward RINm5F cells contrasted with a high rate of the active fraction G75-TIA (gel filtration chromatography; LC(50) of 0.04 mg/mL).