Rat pancreatic elastase was purified on a column of lima bean trypsin inhibitor-Sepharose after inactivating trypsin and chymotrypsin in the pancreatic homogenate by treatment with N^α-p-tosyl-L-lysylchloromethyl ketone and N-tosyl-L-phenylalanylchloromethyl ketone. The molecular weight of the enzyme was 25000, as determined by polyacrylamide gel electrophoresis. A radioimmunoassay method was established for rat elastase ; the enzyme level could be determined over the range of 2.5-300 ng of the enzyme per ml.