Hemolysins were purified from whole bodies of the sea anemone Anthopleura japonica by acetone precipitation, ion-exchange chromatography on CM-cellulose, gel filtration on Sephadex G-75 and chromatofocusing. Two hemolysins (AJH-1 and 2) were detected and only AJH-2 was obtained in pure form as judged by analytical disc electrophoresis. AJH-2 exhibited a lethal activity in mice (LD₃₀ 78μg/kg, i, v. injection) as well as a hemolytic activity (106, 500 HU/mg against sheep erythrocytes). It was stable over a wide range of pH values from 2 to 11 but rather unstable to heat treatment. The molecular weight was determined to be 17, 800 by sedimentation equilibrium or 19, 500 by SDS-polyacrylamide gel electrophoresis, indicating that AJH-2 has no subunit structure. AJH-2 was a slightly basic protein (pI 8.0) and its amino acid composition was characterized by the absence of half-cystine. It was notable that these properties of AJH-2 were closely similar to those of parasitoxin, the major lethal hemolysin isolated from the sea anemone Parasicyonis actinostoloides.