Bordetella Pertussis Tohama phase I was cultured in a 300-liter fermentor using a medium containing 0.1% heptakis (2,5-0-dimethyl) beta-cyclodextrin (MeCD). Pertussis toxin (PT) and filamentous hamagglutinin (FHA) were purified using affinity and ion exchange gel column chromatographies. Endotoxin contents of these antigens (10 micrograms PN/ml) were less than 10 ngLPS/ml. PT and FHA were independently treated with formalin in the presence of amino acid and were mixed at a protein concentration ratio of 1:4, the same ratio of our commercialized acellular pertussis vaccine. PDT vaccine containing 2 micrograms PN of PT and 8 micrograms PN of FHA per milliliter was prepared. This PDT vaccine satisfied all the items of the Japanese Minimum Requirements including potency and toxicity tests. Even after this vaccine was incubated for 4 weeks at 37 degrees C, no deaths of the inoculated mice were observed after challenge with 4 mg of histamine on the 4th and 12th day of the inoculation. Compared with the conventional vaccine, this new vaccine caused less swelling in the mouse footpad test. A field trial of our two vaccines, one manufactured by the conventional method (lot No. 21A) and the other produced by the new method (lot No. KC8702), revealed that children receiving KC8702 showed almost the same anti-PT and anti-FHA antibody levels as those given 21A. Those who received KC8702 suffered from less local side effects such as redness, swelling or induration than those given 21A. Our new method for the production of acellular pertussis vaccine permits us the economical manufacturing of the vaccine with uniform quality in a closed system.