PTA1 (platelet and T cell antigen 1), designated as CD226, is one of the major activating receptors on NK cells. PTA1/CD226 is also broadly expressed on a variety of hematopoietic cells, including Th cells, CTLs, NK T cells, monocytes, DCs, and mast cells, and is involved in multiple immunoregulation. Murine PTA1 (mPTA1) is the homolog molecule of human PTA1. Using mPTA1-Fc as immunogen and the technique of rat B lymphocyte hybridoma, we raised five hybridoma cell lines secreting monoclonal antibodies (MAbs) to mPTA1, designated FMU-mCD226.1 approximately FMU-mCD226.5. Rat immunoglobulin class and subclass of the MAbs FMU-mCD226.1 approximately 5 were determined to be IgG2a, IgM, IgM, IgG1, and IgG1, respectively. FMU-mCD226.2, 3, 4, and 5 worked well in Western blot assay and could also recognize natural PTA1 on the surface of EL-4 cells and mPTA1 cDNA transfected 293T cells detected by FCM. Thus, successful production of rat anti-murine PTA1/CD226 monoclonal antibodies provides a new powerful tool for investigation of murine PTA1 function in the mouse model, both in vitro and in vivo.