DNA was extracted enzymatically from full-thickness intestinal resection specimens from 40 patients with Crohn's Disease (CD) and 40 cancer or diverticular disease controls. 5' IS900 PCR was performed using primers shown to be capable of detecting a single M. paratuberculosis genome. Under optimal conditions 300 bacilli could be detected in 1g of tissue. M. paratuberculosis DNA was detected in 26 of 40 (65%) CD tissues and in 5 of 40 (12.5%) control tissues. Stringent precautions were taken to exclude artefact due to laboratory contamination. The presence of M. paratuberculosis in a small proportion of control tissues is consistent with a previously unrecognized environmental prevalence. Its presence in a majority of CD tissues is consistent with a role for M. paratb in the pathogenesis of this disease.