4-1BB, a member of the tumor necrosis factor receptor (TNFR) superfamily, is a costimulatory receptor that is primarily expressed on activated T cells and professional antigen-presenting cells. In this study, the expression pattern of 4-1BB on immunology cells and tumor cells was explored by flow cytometry using newly generated three anti-4-1BB monoclonal antibodies (mAbs; 6F9, 7D6, and 1G11), which bind to distinct 4-1BB epitopes. Compared with the available 4-1BB mAb 4B4-1 that recognized 4-1BB on activated T cells and monocytes, the novel mAbs also could recognize 4-1BB on some cancer cell lines, particularly on lung cancer cell lines such as SPC-A-1, H446, H460, and H1299 by flow cytometry analysis, western blot, and RT-PCR. Immunohistochemistry staining showed the 4-1BB was expressed on lung tumor tissue (33/35) but not on normal lung tissue (3/3). It was determined that 4-1BB was strictly expressed on lung cancer cells, which may provide information on the 4-1BB signal in tumor immunology mechanism.