Five synthetic peptides identified as antigenic sites on the S1 subunit of pertussis toxin (PT) were coupled to the diphtheria toxin cross-reactive mutant protein CRM 197 or BSA. All peptide conjugates were immunogenic in animals. Two peptide-CRM-conjugates, corresponding to amino acids 1-17 and 169-186, induced especially high antibody titers against native PT in mice (Balb/c, C57/Black and outbred NMRI) as measured by ELISA. Upon in vivo PT challenge (0.5 microgram toxin) of the NMRI mice both the CRM and BSA conjugates of these two peptides fully protected the mice from PT induced leucocytosis. Some of the protected mice receiving peptide 1-17 conjugate had very low antibody titers against PT but high titers against the peptide as measured by ELISA, showing that PT-ELISA does not always reflect in vivo protection. A booster response against PT was noted upon challenge with PT in mice receiving peptide 1-17 or peptide 169-186 conjugate. Antibodies against peptide 170-186 could not be evaluated by ELISA since conjugates of this peptide (like PT itself) bind to immunoglobulins. They may also caused clustering of CHO cells. Rabbit antiserum to the peptide 1-17-CRM conjugate was highly efficient in inhibiting the ADP-ribosylating activity of PT with bovine transducin as substrate whereas the rabbit antiserum raised against the peptide 169-186-CRM conjugate neutralized the clustering effect of PT on CHO cells. Thus there is no concise correlation between the in vivo protection against PT challenge and the in vitro methods used for measuring antibody levels against PT (neutralization of the enzyme activity, the CHO cell clustering activity and titers in PT-ELISA). The CRM-conjugates of these two peptides constitute the first synthetic pertussis vaccine candidate with the ability to provide a chemically well defined, safe and efficient pertussis vaccine.