Mycobacterium paratuberculosis, a slow-growing and mycobactin-dependent mycobacterium, is the etiologic agent of Johne's disease in cattle, and it has been recently associated with Crohn's disease in humans. The infection is acquired early in life, but it has a long incubation period, during which the animals shed the bacterium with the feces, thus contributing to the spread of the infection. Early diagnosis of infection by isolation or antibody detection is hampered by the lack of rapid, specific, and sensitive techniques. Johne's disease is present in our regions, and we are evaluating the use of biomolecular techniques to detect the bacterium. We used primers P11 and P36, described by Moss et al., to set up a PCR and a DIG-labelled probe specific for M. paratuberculosis; the probe was synthesized by a one-step PCR. These primers amplify a 278 bp sequence in the IS900 fragment which is present in multiple copies in the M. paratuberculosis genome. We are investigating a herd of goats likely affected by Johne's disease. We received a resected piece of intestine from one of the diseased animals, and fecal samples from the entire herd. The clinical diagnosis of the diseased animal was confirmed histopathologically. We report the results obtained with the intestine. The finely minced tissue was suspended by Lysing solution and digested with Proteinase K; the DNA was extracted by Chelex treatment, and the supernatant used as target in the PCR. After agarose gel electrophoresis a band of the expected size was seen, and the PCR product dot blotted and hybridized with our DIG-labelled probe showed a strong signal, thus confirming the diagnosis. PCR results were obtained in less than two days. Biomolecular techniques seem particularly suitable for the early diagnosis and for the investigation of the role in humans, and the epidemiology of this fastidious bacterium.