After several decades of study little is known of the response of Mycobacterium avium subspecies paratuberculosis (Mptb) to the hostile environment it encounters within the infected host. In order to address this issue a pilot study of gene expression in the in vivo environment was undertaken. Real Time polymerase chain reaction was used to analyse bacterial mRNA extracted from the infected tissue of animals clinically affected with Johne's disease. Small intestine was rapidly removed surgically, lumenal contents washed away and the mucosa and submucosa scraped off. Mycobacteria in this material were released from the intra-macrophage environment by gently isotonic lysis and recovered for RNA extraction by differential centrifugation. This RNA was compared with RNA extracted from bacteria grown in artificial culture media (so called in vitro sample). Genes selected for study included a number flanking IS900 insertion sites, and others were genes that could potentially be associated with response to the intra-macrophage environment. One gene in particular, katG encoding the catalase/peroxidase, seemed to show an on/off switch between the in vivo and in vitro environment. In addition differential expression of the gene 2 associated with the IS900 insertion locus 13, and gene 1 of locus 10, was observed. A comparison of mRNA samples from cultured Mptb with samples from M. avium subspecies avium indicated a general up-regulation for genes associated with IS900 insertion loci covered in this study. This approach to the identification of disease associated gene expression will be extended with partial (sub-set) genome microarrays.