Current methods for the isolation and identification of Bordetella bronchiseptica from clinical samples are time-consuming and are based, in part, on subjective observations. We describe the use of a Bordetella-specific DNA probe in a nonradioactive colony lift-hybridization assay for the identification of B. bronchiseptica. Eleven of 82 clinical specimens were found to contain B. bronchiseptica by this method, while only 5 of these were reported to contain the organism when the specimens were analyzed by traditional methods. The chromosomal fragment containing a sequence complementary to the probe appeared to be conserved in B. bronchiseptica isolates from swine from a variety of sources. The assay is more rapid than culture and biochemical testing since it can be performed directly on primary culture plates, even when they are heavily contaminated with other bacterial species. Only minimal training is required to accomplish the assay successfully, and the results are easy to interpret.