Background and Aims. PCR methodology has opened a new field in the diagnosis of Johne's disease (JD). The control of JD relies primarily on good management and early detection of infected animals. We reported a relatively simple extraction procedure and a nested PCR (amplification with primers P90 and P91 followed by amplification with primers P11 and P36, specific for sequences within the IS900 fragment) to detect M. paratuberculosis DNA in tissue and fecal samples. The sensitivity of the nested PCR, estimated with a crude extract of the bacterium grown in vitro, was 5fg. Three cows from a herd of 78 animals, had symptoms related to JD. We planned to study the entire herd, and to compare the routine tests with PCR. Materials and Methods. Fecal and serum samples were obtained from the entire herd, and tissue samples from the diseased animals only. An aliquot of the tissue and fecal samples was processed for routine investigations (AFB staining, isolation), and the remaining was stored frozen until PCR processing, to be carried out blindly. The specimens were treated with Proteinase K and SDS, then boiled with Chelex. The diluted supernatant was directly used for the amplification. Results and Conclusions. Of the entire lot of fecal samples, 5 were AFB positive; isolation and serologic results are not yet available. Out of 34 samples so far studied by PCR, 12 were positive. The relatively simple extraction procedure might be exported to routine diagnostic laboratories, and together with the nested PCR might help to speed up the diagnosis of JD.