The characteristics of hemolytic action of cytolysin produced from V. vulnificus were investigated in mouse erythrocytes. The cytolysin bound erythrocyte membranes in temperature-independent manner and then lysed cells temperature-dependently. Hemoglobin release by the cytolysin was completely inhibited by the presence of raffinose or melezitose, but K+ release was not affected. The cytolysin-induced hemolysis was always accompanied with the conversion of membrane-bound cytolysin into an oligomer of 210 kDa, corresponding to a tetramer of native cytolysins. Nonesterified cholesterol inactivated the cytolysin by converting active monomeric cytolysin into inactive oligomer. The results suggest that the cytolysin lyses erythrocytes due to the formation of small pores on erythrocyte membrane by cholesterol-mediated oligomerization of the cytolysin.