Pathogenic strains of Bordetella pertussis undergo spontaneous phase variation and become non-pathogenic upon culturing in vitro. The spontaneous process was studied in pathogenic B. pertussis strains Tohama, 165 and 18323 by isolating spontaneous variants, selected for their ability to grow on synthetic and semi-synthetic solid media. In strains Tohama and 165, the frequency of variants able to grow on synthetic and semi-synthetic media was between 10(-6) and 10(-7). About 250 variant strains were screened for the presence of virulence-associated traits, such as production of hemolysin, pertussis toxin and filamentous hemagglutinin (FHA). Only four different combinations of the traits were found: 7-11% of the variants displayed all traits, 17% of the variants carried pertussis toxin and FHA, 5-11% carried FHA only and 66% were devoid of all virulence traits. The strains which had at least one virulence trait also demonstrated some adenylate cyclase activity. The disappearance of hemolysin was related quantitatively to the other traits. These results suggest that phase variation in B. pertussis is a non random process, involving ordered disappearance of virulence factors in the following order: hemolysin, pertussis toxin and FHA. Since all the variant strains were phenotypically stable upon further passaging in vitro, they represent the stable, final outcome of the variation process which may have occurred in "Phase I" colonies. In contrast, 300 variants of B. pertussis 18323, which were able to grow on selective solid media, carried all the virulence traits. This is in accordance with the strain's unique intracerebral virulence.