Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) enters intestinal epithelial cells of cattle and other ruminants via a mechanism that remains to be fully elucidated. In this study, we observed that the M. paratuberculosis 35-kDa protein, also termed major membrane protein (MMP), plays a role in invasion of bovine epithelial cells. The gene encoding the major membrane protein (MMP) was cloned and expressed as a fusion protein with the maltose binding protein (MBP/MMP) in E. coli. Rabbit antisera were raised against a M. paratuberculosis whole cell sonicate and MMP-specific antibodies were purified from rabbit sera by affinity chromatography. Immunoelectron microscopy of M. paratuberculosis bacilli labeled with MMP-specific antibodies shows that the protein is localized to the surface of these bacteria. Cattle with Johne's disease produced antibodies against MMP but did not produce IFN-g, suggesting the protein elicits a humoral but not cell-mediated immune response. Both anti-MMP antibodies and MBP/MMP protein inhibited M. paratuberculosis invasion of cultured MDBK cells by 30%. Similar invasion experiments with M. paratuberculosis incubated in low oxygen tension, a condition simulating in the intestine, decreased invasion by 60%. Collectively, these data show that the 35-kDa protein is a surface exposed protein that plays a role in invasion of epithelial cells. From these studies, we suggest that the major membrane protein is a virulence factor of M. paratuberculosis that may be important in the initiation of infection in vivo.