Some properties of he nolysin produced by Vibrio vulnificus were investigated. The hemolysin was heat labile, and the hemolytic activity was inhibited by adding cholesterol or divalent cations. Cholesterol inhibited the temperature-independent hemolysin-binding step, suggesting that cholesterol made up the binding site. of the cell membrane, whereas the divalent cations inhibited the temperature-dependent membrane-degradation step. However, the V. vulnificus hemolysin was stable to oxygen and sulfhydryl reagents and was not inactivated by antiserum against streptolysin O, suggesting that the V. vulnificus hemolysin differs from oxygen-labile hemolysins which bind to cholesterol. The V. vulnificus hemolysin seems to be one of the exceptional cholesterol-binding hemolysins.