Extracts of the body walls of the adult Ascaris lumbricoides var. suis inhibit pepsin between pH 1 and 6. Four inhibitors of pepsin were isolated and purified as follows. The crude extract of Ascaris was incubated at 37° and pH 2.0 for 75 min. The pepsin-inhibiting activity was obtained in a fraction precipitated at 0.65 saturation with ammonium sulfate at pH 5.35, and sequentially chromatographed on BioGel P-30 at pH 2.1 and Cellex SE at pH 4.7 and resolved into four inhibitors on DEAE-Sephadex at pH 8.8. The degree of purity of each inhibitor was demonstrated by disc gel electrophoresis. The over-all yield of pepsin inhibitors was 44% of the initial extract. Inhibitor I was 7%; Inhibitor II, 10%; Inhibitor III, 20%; and Inhibitor IV, 7%. The over-all purification of Inhibitors I → III was 6,800-fold and that of Inhibitor IV, 3,300-fold. The molecular weights of these inhibitors were calculated from their amino acid composition and were in agreement with the values obtained from both 5% and 10% polyacrylamide gels in sodium dodecyl sulfate. The values obtained from the amino acid composition were: 17,515 (= 160 amino acid residues), 15,584 (= 142 amino acid residues), 16,124 (= 147 amino acid residues), and 31,719 (= 290 amino acid residues) for Inhibitors I, II, III, and IV, respectively. The NH2-terminal amino acid residue of each of these proteins was histidine. The ability of each of these inhibitors to inactivate pepsin was lost by its treatment with either trypsin or chymotrypsin. Each inhibitor inhibited porcine, bovine, and human pepsins, and porcine gastricsin, but not human gastricsin.