Although Mycobacterium paratuberculosis has been reported by both culture and polymerase chain reaction in unfixed intestinal tissue from patients with Crohn's disease, its pathologic significance is unclear. We used polymerase chain reaction to analyze DNA for the presence of M. paratuberculosis from 27 specimens in 23 patients with Crohn's disease. Seventeen of these cases contained granulomata. DNA from unstained paraffin tissue sections was amplified using nested PCR specific for a 264-base pair fragment of IS900, a multicopy insertion element specific to M. paratuberculosis. The sensitivity of this technique was demonstrated by the amplification of small numbers of cultured organisms in a paraffin cell block in the presence of large amounts of added human DNA. M. paratuberculosis was not identified in any of the specimens from patients with Crohn's disease, nor in any of 11 normal colon sections tested. Therefore, we were unable to confirm a role for M. paratuberculosis in Crohn's disease.