α1-Antitrypsin was isolated as an electrophoretically homogeneous protein in preparative quantities from malignant human breast tissue by a two-step procedure of affinity chromatography on Sepharose-chymotrypsin, followed by polyacrylamide gel electrophoresis. Additionally, α2-macroglobulin and α1-antichymotrypsin were bound to, and subsequently eluted from the column. Antithrombin III and α1-acid glycoprotein, which were also components of the tissue extracts, were not bound, and appeared in the breakthrough peak. α1-Antitrypsin appeared to be partially modified immunologically by Sepharose-chymotryppin chromatography. However, α2-macroglobulin remained immunologically unaltered. Upon subsequent polyacrylamide gel electrophoresis, the α1-antitrypsin regained its capacity to give an immunological reaction of complete identity. Inhibitory activity toward proteases is also observed in filtrates of 1000 molecular weight cut-off membrane filters. Endogenous caseinolytic activity was observed in several eluate fractions. However, only N-benzoyl-L-tyrosine-ethyl ester, of the model compounds tested, was cleaved. The esterase activity is attributed to a breast tissue component since the column was free from dissociable chymotrypsin. Affinity chromatography on Sepharose-chymotrypsin represents a means of rapidly separating 37.5% of the total antichymotryptic activity from breast tissue extracts in high purity, in a single step.