We have isolated 120 mutant strains producing pertussis toxin (PT) cross reacting materials (CRMs) from B. pertussis, strain Tohama, phase I by nitrosoguanidine treatment. Strains producing higher PT tend to show higher virulence in mice. No direct correlation between the virulence and other factors, such as filamentous hemagglutinin, adenylate cyclase or dermonecrotic heat labile toxin, was found. Most CRMs were less reactive to the anti-S1 monoclonal antibody, 1B7. When the PT CRMs produced by strains 69D, 74E or 79G, which were less or non-toxic, were mixed with A protomer purified from native PT, the PT activity assayed by clustering of CHO-cells increased significantly, but not when they were mixed with B oligomer. These CRMs may be composed of defective S1 and intact S2, S3, S4 and S5. Molecular sizes of PT CRMs outside and inside the cells were analysed by sucrose density gradient centrifugation. The sizes of the CRMs were in the range of 10K to 210K, but the biological activity of PT was detected at only the same molecular size, 106 K, as native PT. The majority of the CRM was released into culture medium if all five subunits were assembled; otherwise they accumulated inside the cell without completion of assembly to form the hexamer in the PT-form. One of the non-toxic mutants named 79G showed one point mutation from G to A at the 730th base from the Eco R1 site of the PT gene. Replacement of Cys-41 with Tyr-41 in S1 must have resulted from this mutation. 79G PT composed of S234 (5) was accumulated both inside and outside the cells because the mutant S1 could not form the disulfide bond in the molecule to form the hexamer with the B oligomer, and also S1 must be degraded because of its instability in the cells. Nevertheless 79 GPT showed high immunoprotectivity in mice by active or passive immunization against ic or aerosol challenge with B. pertussis, strain 18323, respectively. It may have a proper conformational structure for protective immunogenicity and could become a good candidate strain for production of a safer and effective pertussis vaccine in the future.