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A panel of neutralizing murine monoclonal antibodies (MAbs) against Staphylococcus aureus alpha-toxin has been established, using formaline-inactivated alpha-toxin as an immunogen. Five independent groups of neutralizing epitopes have been identified representing five functionally important structures in the toxin molecule. Because none of the antibodies binds to overlapping decapeptides representing the toxin sequence or to bromocyanogen cleavage products of alpha-toxin, they may all bind to conformational epitopes. Nevertheless, they all bind to monomeric alpha-toxin in a Western blot. Three of the antibodies bind to the toxin monomer in an enzyme-linked immunosorbent assay (ELISA) in the presence, but not in the absence, of detergent. These epitopes are not accessible in hexameric toxin; two of them may represent the contact sites of the toxin monomers upon hexamerization and one is related to a structurally important glycine-rich central hinge region. Two different antibodies bind to monomeric toxin in an ELISA in the presence and absence of detergent and their epitopes are present more than once on oligomeric toxin; they bind strongly to hexameric toxin in a Western blot. The binding properties of the antibodies to alpha-toxin in different assay systems are summarized in an epitope model, which describes the presence of neutralizing domains in the different conformational steps required for pore formation.