The aim of this study was to produce monoclonal antibodies (MAbs) to in vivo expressed antigens of Mycobacterium avium subsp. paratuberculosis (Map) that would be useful for the development of assays for the detection of the organism in both disease and food specimens. Eight BALB/C mice were injected with live Map to cause infection. The intention was to stimulate an immune response that would include novel antibodies to in vivo expressed antigens that would be absent from mice immunised with culture-grown dead cells. Hybridoma fusions produced from the spleen cells of these Map-infected mice were screened with antigen derived from the intestines of animals naturally infected with Map. Several hybridomas were selected on the basis that they reacted by ELISA to the Map-infected bovine tissue antigen but not to that from normal tissue. Subsequent testing of the cloned lines failed to differentiate between additional antigens derived from healthy and Map-infected bovine small intestine. In addition, examination of the livers from the infected mice showed little or no sign of granulomatous lesions that would have been indicative of Map infection. Therefore no Map-specific MAbs were obtained. Since there was little evidence to demonstrate that Map mouse infection had occurred, this failure cannot necessarily be attributed to the scientific approach. Mouse infection, demonstrated by granulomatous liver lesions, had been observed in previous experiments, and the reason for failure in this experiment is unclear. The approach towards obtaining MAbs to Map-specific in vivo expressed antigens will continue by attempting to achieve such expression in Map cells cultured in macrophage.