The septin family of GTPase proteins has been shown to be important for cell division, cytoskeletal organization, and membrane-remodeling events. Septin 9 (SEPT9) is a member of the septin family (also designated MSF/eseptin/Sint1) and has been implicated in tumorigenesis. The present study reports on the preparation and properties of a monoclonal antibody (MAb) directed against SEPT9. The antibody was produced by hybridization of mouse myeloma cells with lymph node cells from an immunized rat. The MAb 7B5 specifically recognized SEPT9, as evidenced by immunoblotting using a variety of extracts from cultured cells. In immunostaining using MAb 7B5, a filamentous pattern near the plasma membrane was observed. The MAb 7B5 promises to be useful in immunoblotting and immunostaining experiments in various cells and tissues to determine the expression levels of SEPT9, as well as to further the analysis of the biological function of this protein.