Spheroplast form of M. avium subsp. paratuberculosis (M. para) has been implicated in the pathogenesis of Crohn's disease (CD) and sarcoidosis. Its detection deep in patient tissues would support its association with human disease. However, distinction between the acid-fast and the cultured spheroplast forms in tissue of CD and sarcoidosis patients infected with M. para is difficult. To help identify spheroplast forms in inflamed tissues of patients, a non-radioactive in situ hybridisation method was developed and optimised. We have shown that by in situ hybridisation with the IS900-specific probe we can detect nucleic acids from M. para spheroplasts, but not their acid-fast forms in tissue sections. Our in situ hybridisation assay is proven specific by the negative finding with control tissue preparations containing spheroplasts of related Mycobacteria (M. tuberculosis and M. smegmatis) and unrelated organisms (Helicobacter pylori or E. coli). This novel in situ hybridisation procedure will for the first time provide a way to distinguish between acid-fast and spheroplast forms of M. para and to localise them in tissues. Detection of M. para variants deep in patient's tissue would be an enormous step forward in the search for an etiological agent in CD.