The killing of Escherichia coli by isolated human myeloperoxidase plus hydrogen peroxide plus chloride ions was shown to proceed via an increased permeability of the bacterial cell wall. A correlation between the extent of the increased permeability and the number of surviving colony-forming units was found (P less than 0.0005). The same phenomenon was observed with isolated human neutrophils. The permeability increase was shown to be limited, because low-molecular-weight substrate became freely permeant, but the bacteria retained their permeability barrier for protein. Furthermore, disruption of the permeability barrier was followed by destruction of cytoplasmic protein. The active antibacterial agent was probably hypochlorous acid, the direct product of the system, rather than singlet oxygen, the nonenzymic product of hypochlorous acid and hydrogen peroxide. This is concluded from the fact that the myeloperoxidase system could be mimicked by hypochlorous acid, whereas conditions that favor formation of singlet oxygen did not enhance this effect. The relevance of the system for killing of bacteria at neutral pH is discussed.