M. paratuberculosis has been considered as a putative causative factor of Crohn's disease. However, its detection in diseased tissue samples using the polymerase chain reaction yielded conflicting results. We validated this technique for the detection of mycobacteria (any species) and M. paratuberculosis before applying it to 72 intestinal biopsies from patients with Crohn's disease (N = 36), ulcerative colitis (N = 13), and control subjects (N = 23). Possible polymerase chain reaction inhibitors were detected by spiking template DNA with the equivalent of two M. paratuberculosis genomes. Mycobacteria were found in 17/36 (47%), 6/13 (46%), and 13/23 (57%) tissue samples of Crohn's disease, ulcerative colitis, and controls, respectively. No M. paratuberculosis were detected in any sample. It is concluded that mycobacteria are present with a similar frequency in the intestinal tissues or luminal inclusions of patients with inflammatory bowel disease and of those unaffected by the disease. Our data do not support a role for M. paratuberculosis in Crohn's disease.