Murine monoclonal antibodies were generated against streptolysin O. One out of 10 tested immunoglobulin clones exhibited strong neutralizing activity; in solution, the presence of approximately two to four antibody molecules per toxin monomer effected 50% neutralization of hemolytic toxin activity. An enzyme-linked immunosorbent assay performed with target cell membranes that were treated with streptolysin O in the presence and absence of neutralizing antibodies showed that the antibodies did not block primary binding of the toxin to the cells. When membranes were solubilized in deoxycholate detergent and centrifuged in sucrose density gradients, those lysed with streptolysin O contained detergent-resistant, high-molecular-weight oligomers identical to the pore lesions, whereas those given toxin and neutralizing antibody contained the toxin exclusively in low-molecular-weight, nonoligomerized form. The process of pore formation by streptolysin O must thus involve two distinct steps, i.e., the primary binding of toxin molecules to the membrane followed by oligomerization of bound toxin monomers by lateral aggregation in the lipid bilayer to form the transmembrane pores.