Histological examination in culture of tissues is commonly used to assess the accuracy of other diagnostic techniques and to confirm the diagnosis of Johne's disease in cattle slaughtered in control programs. In cattle with clinical disease due to M. paratuberculosis infection, granulomatous lesions with plentiful numbers of acid fast bacilli are readily observed in the intestinal mucosa and/or the mesenteric lymph nodes, particularly in the ileocecal region. During the subclinical phase of infection, lesions of Johne's disease are less developed, more localized and organisms may be difficult to detect. In a control program on 25 Victorian dairy herds with Johne's disease, tissues from cattle with a reaction to an absorbed ELISA were examined by culture and histology. Routine histological examination by pathologists experienced with clinical Johne's disease frequently failed to identify subclinical Johne's disease lesions which were detected by further prolonged examination of tissues and confirmed by culture. Very small numbers of acid fast organisms and concurrent parasitic and bacterial enteritis contributed to the failure to identify lesions of Johne's disease. Electron microscopic examination of granulomatous lesions in which acid fast organisms were difficult to detect revealed bacilli with deficient cell walls in both macrophages and giant cells. The significance of these findings with respect to the pathogenesis of the disease and for disease control will be discussed. The findings indicate that in cattle with reactions in the absorbed ELISA or where subclinical infections with M. paratuberculosis are suspected stringent histological examination is required. Granulomatous reactions and lesions in which acid fast bacilli are not observed may be due to Johne's disease rather than parasites.