Investigations of M. paratuberculosis, the causative agent of Johne's disease (JD) to date have focussed on bacteria cultured in the laboratory. We report here a method for the isolation of M. paratuberculosis from intestinal sections from cattle and goats with clinical signs of JD. The method utilises characteristics of the mycobacterial cell wall to separate mycobacteria from host tissue for proteomic analysis. Current methods for RNA/DNA extraction from in vivo derived bacteria do not require the high level of purification necessary for proteomic analysis. Moreover, these methods also use reagents that can remove membrane bound proteins, thus altering the proteome of the sample.Sections of the ileum and jejunum from cows (naturally infected) and goats (experimentally infected with M. paratuberculosis) were homogenised in a cold hypertonic buffer and subjected to sonication to lyse eukaryotic cells and other gut microflora. Liberated acid-fast bacilli (identified by ZN stain), were isolated from homogenates by Percoll-assisted differential centrifugation. The purity of the extracted M. paratuberculosis was then assessed using a combination of PCR, ZN staining and two-dimensional electrophoresis (2DE) with reference to an in vitro grown strain of M. paratuberculosis. By extracting M. paratuberculosis in this fashion we have been able to investigate proteomic differences between in vivo and in vitro- derived organisms. This method is the first to provide an opportunity to isolate intact bacteria from the infected host for proteomic analysis and may assist in the characterisation of antigens and potential virulence determinants, expressed specifically by the pathogen in association with the disease process.