We have produced a panel of monoclonal antibodies to pneumolysin, the membrane-damaging toxin from Streptococcus pneumoniae. We have used these antibodies to identify three regions of the toxin sequence that are involved in the lytic mechanism of this toxin. Two of these sites probably form the cell binding site of this toxin. Antibodies to the third site inhibit the lytic action of this toxin but not the binding of this toxin to cells. This site is engaged in the oligomerization process involved in the formation of pores in cell membranes. Two of these epitopes are also present in the related toxin perfringolysin O.