A role of Mycobacterium paratuberculosis in Crohn's disease has been suggested for many years. To determine whether Mycobacterium paratuberculosis has the potential to cause Crohn's disease, we investigated Crohn's disease tissues for the presence of Mycobacterium paratuberculosis, using highly sensitive nested polymerase chain reaction (PCR). DNA was extracted from samples of intestine obtained from 13 patients with Crohn's disease, 14 patients with ulcerative colitis, and 13 control patients without inflammatory bowel disease. Nested PCR was performed using primer pairs complementary to sequences in the insertion element IS900 gene of Mycobacterium paratuberculosis. No Mycobacterium paratuberclulosis-specific PCR product was detected in any of these specimens, although PCR products of the appropriate size were consistently obtained using control DNA template from Mycobacterium paratuberculosis. The results did not suggest that Mycobacterium paratuberculosis is involved in the pathogenesis of Crohn's disease.