Coagreggation of Treponema denticola with either Porphyromonas gingivalis or Fusobacterium nucleatum was characterized and the role of the major outer sheath protein (MSP) in the coaggregation process of these bacteria was evaluated. The MSP of T. denticola was found to be able to bind to P. gingivalis and F. nucleatum cells and this binding could be inhibited by MSP in a concentration-dependent manner. While sodium dodecyl sulfate polyacrylamide gel electrophoresis and Periodic acid-Schiff (PAS) staining of MSP revealed that it is a glycoprotein, monosaccharide analysis showed that MSP contains: Glc (44.4), Gal (20.4%) GlcN (1.3%), GalN (31.6%) and Fuc (9.2%). Peptide N-glycosidase F deglycosylation of MSP was found to inhibit its binding to F. nucleatum but not to P. gingivalis cells. Sugar-binding studies showed that the requirements for the binding of both T. denticola and MSP to F. nucleatum cells are similar to those of the F. nucleatum galactose-binding lectin. These data suggest that MSP acts as an adhesin during the coaggregation process of T. denticola with P. gingivalis and F. nucleatum through its protein and carbohydrate moieties, respectively.