A new crystalline protein, chymo-trypsinogen, has been isolated from acid extracts of fresh cattle pancreas. This protein is not an enzyme but is transformed by minute amounts of trypsin into an active proteolytic enzyme called chymo-trypsin. The chymo-trypsin has also been obtained in crystalline form. The chymo-trypsinogen cannot be activated by enterokinase, pepsin, inactive trypsin, or calcium chloride. There is an extremely slow spontaneous activation upon standing in solution. The activation of chymo-trypsinogen by trypsin follows the course of a monomolecular reaction the velocity constant of which is proportional to the trypsin concentration and independent of the chymotrypsinogen concentration. The rate of activation is a maximum at pH 7.0-8.0. Activation is accompanied by an increase of six primary amino groups per mole but no split products could be found, indicating that the activation consists in an intramolecular rearrangement. There is a slight change in optical activity but no change in molecular weight. The physical and chemical properties of both proteins are constant through a series of fractional crystallizations. The activity of chymo-trypsin decreases in proportion to the destruction of the native protein by pepsin digestion or denaturation by heat or acid. Chymo-trypsin has powerful milk-clotting power but does not clot blood plasma and differs qualitatively in this respect from the crystalline trypsin previously reported. It hydrolyzes sturin, casein, gelatin, and hemoglobin more slowly than does crystalline trypsin but the hydrolysis of casein is carried much further. The hydrolysis takes place at different linkages from those attacked by trypsin. The optimum pH for the digestion of casein is about 8.0-9.0. It does not hydrolyze any of a series of dipeptides or polypeptides tested. Several chemical and physical properties of both proteins have been determined.