A large scale method for preparation of antithrombin III (AT III) concentrate from plasma or from Cohn fraction IV-1 (Fr. IV-1) has been described. It consists of the following steps: (a) partial purification by precipitation of impurities with 20% polyethylene glycol (PEG) 4000; (b) isolation of AT III from the PEG supernatant by batch adsorption and elution on heparin-Sepharose at a ratio corresponding to 45 vol of plasma or 80 vol of 10% Fr. IV-1 solution to 1 vol of gel; (c) concentration and desalting of the eluted AT III on a Pellicon ultrafiltration system; (d) pasteurization of AT III concentrate by heating for 10 h at 60 degrees C in the presence of 0.5 M sodium citrate at pH 7.5; (e) removal of excess citrate by gel filtration on Sephadex G-50; and (f) sterile filtration, filling and lyophilization. The recovery by activity was 32% from a 113-liter plasma batch and 16% from a 42-kg Fr. IV-1 batch. Both AT III concentrates, derived either from plasma or from Fr. IV-1, had similar specific activity and electrophoretic purity, were nonpyrogenic and met all other FDA requirements for biologic products. Pasteurization induced changes in disc gel and isotachophoretic patterns of AT III preparations.