To clarify the etiologic significance of Mycobacterium paratuberculosis in Crohn's disease, we investigated whether M. paratuberculosis was detected in intestinal tissues, including Peyer's patches, where M. paratuberculosis invades, and colonic lymph follicles, where early lesions appear. Fifty-one samples of intestinal tissues, either therapeutically resected or biopsied, including 34 specimens from 30 patients with Crohn's disease, were studied. Four Peyer's patches and eight lymph follicles were included in the intestinal tissue samples of Crohn's disease. They were visualized by acetic acid fixation. DNA extracted from intestinal tissues by proteinase K treatment was used for nested polymerase chain reaction (PCR) for detection of IS900, which is specific for M. paratuberculosis. PCR products were analyzed by agarose gel electrophoresis and subsequent Southern blot analysis. Our amplification system could detect 7.5 fg of M. paratuberculosis DNA. None of the tissue samples showed positive IS900 amplification, whereas they all showed amplification of the positive control human leukocyte antigen (HLA)-DQA DNA. Spiked experiments of tissue samples with M. paratuberculosis demonstrated that inhibitors of IS900 amplification were not present in the samples. Our study does not support the etiologic significance of M. paratuberculosis in Crohn's disease.