Mycobacterium avium spp. paratuberculosis (MAP) causes chronic granulomatous inflammation of the intestinal tract in many species of animals, but the mechanisms of disease are poorly understood. Attachment of bacteria to epithelial cells is a critical step in pathogenesis of many mucosal diseases. The goal of these studies was to develop an in vitro method to study attachment of MAP to bovine intestinal epithelial cells. Short-term, bovine intestinal organ cultures were used to show a significant difference in the ability of radiolabelled MAP strains to attach to intestinal epithelium. We found significant differences in the ability of different strains of MAP to attach, but there were no differences in attachment among different regions of the intestinal tract. Examination of acid fast stained tissue sections of organ cultures demonstrated that organisms were located adjacent to mucosal epithelium or within goblet cells. Coating of the organisms with fibronectin, which has been shown to be involved in attachment of many mycobacteria, including MAP, affected the attachment of the MAP strains in different ways, but did not affect the overall attachment of the organisms to different regions of the gastrointestinal tract. This organ culture method should also prove useful for defining the molecular mechanisms of attachment and interactions of MAP with intestinal epithelium.