The genetic relatedness of the three medically important Bordetella 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 Bordetella pertussis, Bordetella parapertussis, and Bordetella 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 hands 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.