Objective To establish a nest PCR method and its application for testing 16S rDNA of Clostridium Piliforme. Method The nest PCR condition was modified on the basis of Goto's report. Results The outside pair of primers can lead to the synthesis of a 625*!bp DNA fragment while the inside pair of primers can lead to a 196 bp DNA fragment. Both amplified fragments are exactly the same size as Goto 's report. The 196bp DNA fragment has been demonstrated to be the specific sequence conserved in Clostridium Piliforme in our experiment. Our PCR assay is of great sensitivity, detecting even 2 organisms in positive sample. The sequence of 625 bp DNA fragment amplified in our positive control is 99% homologous to both the MSK and RJ strains as reported by Goto. Conclusion The nest PCR assay established in our laboratory can be used to amplify specifically 16S rDNA of Clostridium Piliforme. 5 liver samples each from mouse, rat, golden hamster, 10 liver samples each from gerbile, guinea pig bred in conventional environment and 1 liver sample infected with MHV3 from mouse bred in clean environment have been tested for Clostridium Piliforme by this method. No latently infected animals have been identified among the 36 samples.