Mycoplasma pulmonis was specifically detected by using a 2.3 kilobase pair (kbp) cloned DNA fragment derived from M. pulmonis m 53 as a probe. This probe recognized 2.3-kbp DNA fragments of three M. pulmonis strains in Southern hybridization, while it did not hybridize with the DNA of M. arthritidis or M. neurolyticum. Determination of the sensitivity of the probe by dot hybridization revealed that 10 ng of M. pulmonis DNA was detected by a biotinylated probe and 1 ng of M. pulmonis DNA was detected by a radioactive probe.