The cilia-associated respiratory (CAR) bacillus is an unclassified, gram-negative, motile bacterium that has been implicated as an etiologic agent of respiratory disease in laboratory rodents. In the present study, approximately 1,200 bases of the 16S rRNA gene from three CAR bacillus isolates were sequenced. CAR bacillus-specific primers were designed on the basis of the 16S rRNA gene sequence and used in a PCR assay. The PCR assay detected as little as 500 fg of purified CAR bacillus DNA. The expected 267-bp DNA fragment was amplified from respiratory tissue of frozen, formalin-fixed, and paraffin-embedded samples from experimentally and naturally infected rats and mice. In contrast, no product was amplified from respiratory tissues of sham-infected experimental animals or animals that were serologically or histopathologically negative for the CAR bacillus. Our findings indicate that this PCR assay is a rapid, specific, and sensitive detection method for the diagnosis of CAR bacillus infection in rats and mice.