To assess the molecular mechanisms involved in cell-cell interactions of T cells, we produced a mAb that could block PMA-induced homotypic T cell aggregation. The established mAb (TN14 mAb) showed an inhibitory effect on T cell aggregation as large as that of mAb against lymphocyte function-associated Ag-1. TN14 mAb showed a strong reactivity with cell surface molecules on T cells, whereas it did not react with any cell surface Ag on macrophages, B cells, bone marrow cells, or WEHI 164 cells, which were used for immunization. However, all cell lysates could react with TN14 mAb in Western blotting analysis, indicating that TN14 mAb recognized some cytoplasmic protein. From biochemical analysis, TN14 mAb was demonstrated to react with the 43-kDa cytoskeletal protein actin. Moreover, from an immunoprecipitation study, TN14 mAb was demonstrated to cross-react with the Thy-1 molecule on T cells. The binding activity of TN14 mAb with the Thy-1 molecule on T cells was strongly blocked by the addition of purified actin, indicating that TN14 mAb recognized an actin-related epitope of the Thy-1 molecule. TN14 mAb blocked not only T cell aggregation but also cell-mediated cytotoxicity by CTL. These results strongly support the hypothesis that the Thy-1 molecule, a member of the Ig superfamily, may play an important role in cell-cell interactions of T cells.