Abstract: A process is provided for the purification of a proteinaceous physiologically active substance having antitumor activity, which is induced by administering to a rabbit at least one substance having a capacity for stimulating reticuloendothelial system and then injecting endotroxin from a Gram-negative bacterium into the rabbit. The process comprises contacting a crude solution of said proteinaceous physiologically active substance with a basic anion exchanger to have said physiologically active substance adsorbed on the anion exchanger, eluting the adsorbed physiologically active substance, and subjecting the eluate containing said physiologically active substance to gel filtration with a gel suitable for separation of a substance with a molecular weight in the range of 30,000 to 70,000. The purified preparation of said physiologically active substance thus obtained may be used as an antitumore agent for the treatment of malignant tumors.