The diagnosis of infection with Mycobacterium avium subsp. paratuberculosis was studied in 12 cattle from infected dairy herds and four from non-infected herds. A comparison was made of (1) histopathological examination and bacterial culture of tissues, (2) culture of serial samples of rectal contents, and (3) examination of repeated blood samples for interferon-gamma (IFN-gamma) and antibody responses. Tissue samples were taken from the small and large intestine and corresponding mesenteric lymph nodes, and from the pharyngeal tonsil and other lymphoid nodes (retropharyngeal, mediastinal, hepatic and supramammary). Histopathological examination and bacterial culture of tissues each revealed that six of the 16 cattle were infected, whereas repeated culture of rectal contents revealed only five infected animals. Except for the hepatic lymph node of a single animal, culture-positive tissues were confined to the intestinal tract and related lymph nodes. Bacterial culture of tissues from the ileum, caecum and lymph nodes draining the jejunum revealed the highest number of infected animals. Moreover, slightly greater numbers of positive tissues were revealed by culture than by histopathological examination. For both the IFN-gamma response and the antibody response, the means of the values for the final three samples before slaughter were significantly higher in infected than in non-infected cattle. However, these immunological responses were too variable to provide a reliable indication of infection.