Pertussis toxin, the most important protective antigen of Bordetella pertussis, is a 106-kDa hexameric protein composed of an A-protomer (subunit S1) and a pentameric B-oligomer (S2 + S3 + 2S4 + S5). The most potent mouse-protective monoclonal antibodies against both respiratory and intracerebral infections were specified for either S1 or S4 and competed with each other in binding to epitopes of native pertussis toxin captured by haptoglobin or in solution, although they did not compete on unfolded pertussis toxin. These data suggest that the protective epitope(s) of S1 and S4 are very closely correlated; they are probably close together sterically. Non-protective anti-S1 and anti-S4 monoclonal antibodies recognized inner antigenic determinants which are not exposed on the surface of native pertussis toxin and interfered with association of the A-protomer and the B-oligomer. These data suggest that the A-protomer and the S4 subunit of the B-oligomer may be closely associated in the native hexameric pertussis toxin molecule.