The relationship between mouse-protective activities against aerosol and intracerebral (i.c.) challenge with virulent B. pertussis organisms and pertussis toxin (PT)-neutralizing activities against ADP-ribosylation (ADPR), CHO cell-clustering (CC), leukocytosis-promoting (LP) and islet-activating (IA) activities of anti-PT antibody, was investigated using anti-PT mouse monoclonal antibodies (MAbs): ten anti-S1, four anti-S23, one anti-S2, two anti-S3, and three anti-S4 MAbs. All the anti-S1 MAbs neutralized ADPR activity of PT. Among them, six MAbs: 1B7, 1D7, 3F11, 10D6, 8G4 and E1E, showed mouse-protection in either an aerosol or i.c. challenge system. These protective MAbs neutralized both LP and IA activities but showed little or no neutralization against CC activity except for 1B7. All anti-S2 and/or S3 MAbs showed higher CC-neutralizing activity and protected the mice against the aerosol challenge, but very little against the i.c. challenge. The mouse-protection was not necessarily parallel to either PT-neutralizing activity or CC-neutralizing activity. The protective MAbs against S1, S2 and S3 decreased the number of bacteria and the amount of pertussis toxin in the lungs of mice challenged with the aerosol. These results suggest that the role of the anti-PT antibody in mouse-protection is not only neutralization of PT but also prevention of bacterial growth in the respiratory tract. Four anti-S1 and two anti-S4 MAbs showed neither neutralization nor protection. Competitive ELISA using the biotinylated MAbs suggested that four anti-S1 MAbs: 1B7, 1D7, 3F11 and 10D6, which showed the highest and almost complete mouse-protection, may recognize the same or closely related epitope(s) or areas on PT. The foregoing results suggested that prediction of the protection of MAbs only by the neutralizing activity against any one of the PT activities must be difficult and that competition ELISA may be helpful for such predictions.