A mouse mAb, which recognized a rat T cell surface Ag responsible for the T cell activation, was produced by a regular hybridoma method using F344 rat T cells stimulated with PMA and a calcium ionophore, as the Ag. The mAb termed 1F4 (kappa-IgM) was reactive with rat T cells but not with B cells and immunohistochemically it stained rat thymus tissues strongly at medulla and weakly rat cortex. Addition of 1F4 mAb to a culture of T cells resulted in the proliferation of T cells by a help of PMA or a solid support. 1F4 mAb also caused the modulation of the corresponding Ag but not other T cell markers such as CD5, CD2, and OX-52-defined Ag. The 1F4 mAb immunoprecipitated a cell surface component having an apparent m.w. of 25,000 from rat T cells which could be associated with a disulfide-linked heterodimer (m.w. 92,000) consists of subunits having m.w. of about 52,000 and 43,000. These results strongly suggest that the 1F4 mAb recognizes a rat T cell Ag homologous to the human and mouse CD3.