The eradication of paratuberculosis is in relation with the performance of diagnostic test used. Mycobacterium paratuberculosis grows very slowly. The primary colonies may be expected to appear any time from the sixth to the twelveth week after inoculation. The immunological tests are not sensitive enough. The polymerase chain reaction (PCR) may constitute an alternative to the bacteriological or serological methods. The polymerase chain reaction has been developed, using as target sequence an insertion element of 1,451 base pairs (IS 900), specific for M. paratuberculosis (15-20 copies per genome). Two successions of primers has been chosen and optimized by informatic study. The primers are designed to amplify the sequence in double amplification reaction (Nested PCR) (primers 783 and 477 bp respectively). The test was performed in three stages: (1) extraction of bacterial deoxyribonucleic acid (DNA); (2) amplification of the target DNA by means of thermostable DNA polymerase; (3) detection of the amplified DNA by electrophoresis, confirmed by dot blot assay after hybridization with an internal labeled oligonucleotide of digoxygenin. A procedure was performed to allow the detection of M. paratuberculosis in preserved fecal samples, kept in ethanol at 70% or at -20°C and an other one from pathological samples disrupted by sonic treatment. A simple procedure was also developed to identify M. paratuberculosis from paraffin-embedded tissue sections. This procedure permits the rapid preparation of DNA for PCR from paraffin-embedded samples. Since fewer manipulations are required, it should also reduce the chance of inadvertent contamination of samples by extraneous DNAs. The application of Chelex 100 chelating resin will be helpful for routine treatment of paraffin-embedded samples. The sensitivity and the specificity of methods used, particularly double amplification and hybridization, are discussed by comparing the results obtained by bacterial culture.