Cathepsin B (EC 3.4.22.1) from buffalo spleen was isolated to homogeneity and its molecular weight was determined to be 25 KDa. The enzyme was found to be a glycoprotein having a total carbohydrate content of 7%. The NH2- and COOH-terminal amino acid residues were identified as Leu and Thr, respectively. The specific extinction coefficient, E1%1cm, of the enzyme was determined to be 13.2. The value of intrinsic viscosity and equivalent hydrodynamic radius of the enzyme were calculated to be 3.47 ml/gm and 2.34 nm, respectively. Polyclonal antibodies raised in rabbits were found to cross-react distinctly with the purified buffalo enzyme. Using BANA as substrate, the Km and Vmax values were determined to be 0.93 mM and 5.57 Units/mg, respectively. The buffalo enzyme was also found to be highly active against protein substrates, and the Km values for casein and BSA were measured to be 1.12 and 1.74 microM, respectively.