Pertussis toxin (PT) is the major protective antigen of acellular pertussis vaccine (aP). We have established an optimal culture condition for the growth of B. pertussis and the production of PT in a laboratory scale fermentor. It was found that when the dissolved oxygen in medium was supplied with pure oxygen instead of air, the yield of PT was dramatically increased (i.e. from 2-3 mg/l using air to 8-10 mg/l using pure oxygen). PT was purified by affinity chromatography using hydroxyapatite and fetuin-sepharose columns. SDS-PAGE analysis and CHO cell clustering test showed that the purified PT was comparable to the reference PT in purity and biological activity. The purified PT could be detoxified by formaldehyde (d-PT). The results of CHO cell clustering neutralization assay and ELISA showed that the antibody induced by d-PT in mice was comparable to that induced by PT contained in a commercial DTaP. These results indicated that the immunogenicity of our d-PT was retained after the purification and detoxification procedures.