Two hybridomas producing monoclonal antibodies (MAbs) were prepared by fusing myeloma cells (Sp2/0-Ag14) with mouse spleen cells immunized with purified spirosin from Yersinia enterocolitica SYT-11-72 (YE72). The antibodies produced by them were designated MAbs-S5 and S27. They were IgG2a and IgG1, respectively, both with kappa light chains. MAbs-S5 and S27 reacted specifically with spirosin from YE72. On Western blotting after limited proteolysis with Staphylococcus aureus V8 protease, YE72 spirosin revealed peptide fragments of 35 and 37 kDa reacting markedly with MAb-S5, which suggested the presence of an antigenic determinant on these fragments. By cellular fractionation of YE72 and subsequent EIA and Western blot analysis, spirosome was shown to be present in the cytoplasm of YE72.