Mycobacterium avium subspecies paratuberculosis (MAP) is an intracellular pathogen. The differential expression of MAP genes resident in cells that are chronically infected with MAP could highlight the molecular mechanisms behind the pathogenesis of MAP disease. We constructed a microarray of specific PCR products from a bank of MAP specific genes, mycobacterial housekeeping genes and genes previously associated with mycobacterial pathogenesis. MAP strains were introduced into 75cm³ flask cultures of Acanthamoebae polyphaga, allowed to ingest over 3- 4 days, then treated once with 100mg/ml Amikacin for 2 hours and incubated at RT. Control MAP cultures were grown for the same period at RT in amoebic culture medium only. After 4-8 weeks amoebic cultures were differentially lysed and mRNA extracted from the MAP. Equal quantities of mRNA from extracellular and intracellular MAP cultures were mixed. cDNA was generated using a random primer set, flourescently labelled with either Cy3 or Cy5 and hybridised to microarrays at 65ºC overnight. These were then given a high stringency wash and read using a dual laser 428 scanner. Signals were normalised to 16SrRNA dilution series and ratios of intracellular/extracellular signals read and calculated using the Genespring software. Results show a significant increase in expression of genes immediately downstream of IS900 elements confirming the presence of an active mycobacterial promoter associated with intracellular induced expression of p43 in IS900 and a highly significant increase in expression of genes associated with the GS cassette, the desA1 gene and MAP specific genes associated with IS900 Locus 6. The significance of these genes in MAP pathogenesis will be discussed.