Paratuberculosis (Johne's disease) poses a significant economic problem to beef, dairy and sheep industries in the United Kingdom and worldwide and is caused by Mycobacterium avium subspecies paratuberculosis (Map). Understanding the interaction between host and Map at the molecular level will identify mechanisms of pathogenesis that promote bacterial survival in host tissue. We have used 2-D PAGE as a tool to investigate the virulent state of Map, incorporating the technique of beating the organism with zirconium/silicon beads to provide a comprehensive representation of the proteome of the organism. A direct comparison of the proteomes of Map scraped from the terminal ileum of ovine paratuberculosis cases and the identical strain grown in vitro is presented. These analyses have identified a set of ten proteins whose expression is up-regulated during natural infection: 1-Pyrroline-5-carboxylate Dehydrogenase (RocA), a putative Acyl-CoA Dehydrogenase (FAD E14), 2-Methylcitrate Dehydratase, Arginosuccinate Synthase (Arg G), Universal Stress Protein, 30S Ribosomal Protein S2 (RpsB), Peptidyl-prolyl cis trans isomerase (PpiA), Luciferase-like mono oxygenase, Thiosulphate sulphurtransferase (SseA) and Adenosine-tri-phosphate(ATP)-dependent Clp Protease (ClpB). The relevance of these proteins to the in vivo, virulent state of Map is discussed.