Tannerella forsythensis, one of the important pathogens in periodontal disease, has a typical surface layer (S-layer) consisting of regularly arrayed subunits outside the outer membrane. The S-layer in T. forsythensis is suggested to be associated with haemagglutinating activity, adhesion and invasion of host cells; however, its precise functions have been unknown. ORFs encoding the major S-layer proteins (230 and 270 kDa) of T. forsythensis ATCC 43037, tfsA and tfsB, respectively, following the names in a recent report [Lee, S.-W., Sabet, M., Um, H. S., Yang, L., Kim, H. C. & Zhu, W. (2006). Gene 371, 102-111] were determined. To verify the function of the S-layer proteins, three mutants with tfsA, tfsB, or both deleted were successfully constructed by a PCR-based overlapping method. S-layer proteins were completely lost in the double mutant. The single-deletion mutants appeared to lose one of the 230 and 270 kDa proteins. Thin-section microscopy clearly revealed that the 230 and 270 kDa proteins composed the S-layer. Although the S-layer proteins may be weakly related to haemagglutinating activity, these proteins were highly responsible for adherence to human gingival epithelial cells (Ca9-22) and KB cells. These results suggest that the S-layer proteins in T. forsythensis play an important role in the initiation stage of oral infection including periodontal disease.