Summary Various factors affecting the recovery of tetanus antitoxin from toxin-antitoxin floccules by proteolytic enzyme action have been studied to determine the optimum conditions for the reaction. The highest recovery of antitoxin was obtained by treating floccules obtained from an over-neutralized concentrated toxin-antitoxin mixture with pepsin at pH 3.0 and in the absence of electrolyte. Evidence is presented which possibly explains why antitoxin can be recovered from toxin-antitoxin floccules and not from toxoid-antitoxin floccules by the action of pepsin.