One of two hemorrhagic factors, hemorrhagic factor I (HR-I), in the venom of Agkistrodon halys blomhoffii (Mamushi) was highly purified by column chromatography on DEAE-cellulose followed by vertical zone electrophoresis and gel chromatography on a Sephadex G-100 column. Sedimentation, gel chromatography, polyacrylamide gel electrophoresis, and immunoelectrophoresis indicated a high degree of homogeneity of the purified material. It possessed no detectable activities of enzymes present in the crude venom; activities tested are those of proteinases, phospholipase A's [EC 3. 1. 1. 4], nonspecific phosphatases [EC 3. 1. 3. 1 or 3. 1. 3. 2], arginine ester hydrolases, bradykinin releasing, and clotting enzymes, L-amino-acid oxidase [EC 1. 4. 3. 2], and hyaluronidase [EC 3. 2. 1. 35]. The purified HR-I preparation had a sedimentation coefficient of 6.08S and diffusion coefficient of 5.40×10−7cm2 sec−1. Using the observed values of S20, w, D20, w, and v (assumed as 0.7) in the Svedberg equation, the molecular weight was calculated as 91,000. The molecular weight determined by Archibald's method was 80,000. The apparent isoelectric point was pH 4.70 and the mobility was estimated to be −3.55×10−5cm2 sec−1 volt−1 at pH 8.5. The amino acid composition of the HR-I preparation was examined. High contents of aspartic and glutamic acids, arginine, tyrosine, and half-cystine residues were found. Among its neutral amino acids the content of tyrosine was highest followed by that of half-cystine. HR-I contained about 12 percent of neutral sugars. Thus, HR-I was concluded to be an acidic glycoprotein. The hemorrhagic activity of the purified material was completely neutralized by commercial Mamushi antivenin, and the minimum hemorrhagic dose of HR-I was 0.0012μg. The purified material also had a potent lethal effect on mice (L.D.50=0.36 (0.32–0.45) mg/kg). Thus, the HR-I principle seems to be a major toxin in this venom.