Mycobacterium avium is an intracellular pathogen and a major opportunistic infectious agent observed in patients with acquired immune deficiency syndrome (AIDS). Fibronectin is an extracellular matrix protein and is a virulence factor for several extracellular pathogenic bacteria binding to mucosal surfaces. We investigated the fibronectin (FN)-binding proteins in the culture filtrate of M. avium by two-dimensional electrophoresis (2DE). Proteins in Sauton medium of M. avium after 3 weeks were separated by 2DE. The proteins were blotted onto polyvinylidene difluoride membrane and incubated with FN. FN-binding proteins were detected by Western blotting using anti-FN antibody. FN bound to five spots (33 kDa, 32 kDa, 31 kDa, 30 kDa and 25 kDa). N-terminal amino acids of these were determined. The 33 kDa spot corresponded to antigen 85 (Ag 85) C. The 32 and 31 kDa spots were either Ag 85 A or Ag 85 B. The 30 kDa spot corresponded to Ag 85 B of M. avium . The 25 kDa spot corresponded to MPA51 (M. avium MPB51). Thus, FN bound exclusively to the Ag 85 complex and MPA51.