The genetic relatedness of the three medically important Bordeteh species was examined by macrorestriction digestion of DNA with the rarely cutting enzyme XbaI and resolution by pulsed-field gel electrophoresis. Our data showed that BordeteUa pertussis, Bordetella parapertussis, and BordeteUa bronchiseptica produced species-specific macrorestriction profiles and that there was some variation between different isolates of the same species, Conserved bands at 130 and 155 kb occurred with B. pertussis (130 isolates tested), but the nine variable bands between 200 and 412 kb distinguished 21 types. The 10 isolates of B. parapertussis tested produced up to 11 bands at 118 to 375 kb, 4 of which were variable, giving three types-The eight B. bronchiseptica isolates examined produced up to 16 bands at 118 to 394 kb, 11 of which were variable, giving three types. The results of this work are compared with the results of previous DNA-DNA hybridization and multilocus enzyme electrophoresis studies which suggested that these three species are closely related and should be considered members of the same species.The following three medically important Bordetella species have been described: Bordetella pertussis, which causes whooping cough in humans; Bordetella parapertussis, the agent which can be responsible for a mild whooping coughlike disorder in humans; and Bordetella bronchiseptica, an animal respiratory pathogen which is very rarely isolated from humans. These are serologically related bacteria with similar morphologies, sizes, and staining reactions (3). They can be differentiated on the basis of their cultural characteristics, nutritional requirements, biochemical activities, and ranges of surface antigens (12).For in vitro culture from nasopharyngeal secretions, B. pertussis is grown on a medium such as cephelexin charcoal blood agar, on which it produces small, shiny colonies. B. pertussis is a slowly growing, nonmotile organism that produces oxidase and catalase. The urease test is negative. B. parapertussis grows well on blood agar and nutrient agar and is nonmotile. Urease is produced after 24 h. Catalase is produced, while oxidase is not produced. B. bronchiseptica grows as glistening beta-hemolytic colonies on blood agar. This organism is motile, urease is produced after 4 h, and oxidase and catalase tests are positive. All three species ferment glucose and lactose with acid production (2).DNA-DNA hybridization (7) and multilocus enzyme electrophoresis studies (10) have shown that these three species are closely related. It has been suggested that they should be considered members of a single species. Restriction fragment length polymorphism, generated by frequently cutting enzymes, has been described as a highly discriminatory system for determining phylogenetic relationships among isolates of the same species and related species (14). This technique failed to discriminate among isolates of B. pertussis, B. parapertussk, and B. bronchiseptica when DNAs were cut with EcoRI; the three organisms were indistingu...