A 16S rDNA-based polymerase chain reaction (PCR) method specific for Pasteurella pneumotropica was developed. The PCR product, a 395-base pair DNA fragment, was amplified from P. pneumotropica and not from 42 other bacterial species tested, including four other Pasteurella species and Actinobacillus ureae. The PCR method was used to identify 13 previously isolated strains that had been identified as P. pneumotropica by conventional methods: 12 were confirmed by PCR; one that was PCR-negative was re-examined by biochemical methods and determined to be A. ureae. The PCR detection of P. pneumotropica in nasopharyngeal swab specimens from 121 surveillance animals (15 inbred mice and 5 inbred rats from 20 animal rooms) had a high carrier state in healthy laboratory animals; for example, rat swab specimens were 89.6% (43/48) positive by PCR, 8.3% were positive by the direct culture-biochemical method, and 16.7% were positive by the enrichment culture-biochemical method. The positive rate for mice (21.9% [16/73]) was lower than that for rats.