Protease inhibitors against trypsin were extracted from cowpea seeds, purified, and characterized. After the seed powder was defatted with hexane, the cowpea trypsin inhibitor (CpTI) was extracted with 0.15 M NaCl for 30 min. The crude extracts were then heated at 90°C for 10 min, followed by precipitation with 40-65% saturation ammonium sulfate, by which the protein purity increased approximately 15-fold. The CpTI had approximate 88-fold and 186-fold purification after anion-exchange chromatography (Super-Q) and gel filtration (Sephadex G-200), respectively. A broad band of the purified CpTI on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicates a degree of heterogeneity and partial denaturation of CpTI, having a molecular mass of ∼8000 kD. Multiple peaks between 7451 and 8898 by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy also suggest heterogeneity. The purified CpTI was stable at 90°C for 60 min, pH 5-10, and 0-3.0% of NaCl. The purification method described here can be used to obtain highly purified CpTI for its studies such as risk assessment of CpTI genetically modified foods.