Among bacterial haemolysins, cholesterol-dependent cytolysins (CDCs) produced by various Gram-positive bacteria are known to exhibit a lethal activity in mice. In this study, recombinant CDCs of streptolysin O, pneumolysin, ivanolysin O, listeriolysin O and several listeriolysin O mutants were constructed and the relationship between cytolytic activity and the lethal activity of each recombinant protein in mice was examined. Specific activity for cytolysis was determined by a quantitative haemolytic assay. Each protein was injected intravenously into mice and the lethal activity was evaluated by measuring the time until death of the mice. The four full-length CDC proteins exhibited lethal activity and their activities were highly proportional to their cytolytic activities. Inhibition of haemolytic activity resulted in the loss of lethal activity and non-haemolytic mutants of listeriolysin O did not exhibit any lethal activity. These data clearly indicate that the lethal effect of CDC proteins is dependent on the cytolytic activity.