Thymidylate synthase was identified at the cellular level using anti-thymidylate synthase monoclonal antibody (M-TS-4) developed against HeLa cell line. HeLa cells, 9L rat gliosarcoma cells, and some of human brain tumor cells (medulloblastoma, metastatic brain tumors from lung cancer and osteosarcoma) were cultured in complete medium for 72 hr and fixed with 10% buffered formalin. These were covered with 1:20 dilution of M-TS-4 in Burridge buffer and 1% bovine serum albumin for 4 or 24 hr. After rinsing twice with phosphate-buffered saline solution (PBS), the cell staining was made with avidin-biotin peroxidase complex (ABC). In addition, HeLa cells were exposed to 2 microCi/ml of tritiated thymidine for 30 min, cultured again for 0 to 5 hr, and subjected to autoradiography after M-TS-4 staining with ABC. All cells were stained satisfactorily with ABC except 9L rat gliosarcoma cells. Autoradiography revealed that 38% of the cells were stained with ABC, 28% were labeled with tritiated thymidine, while only 8% of the cells were stained simultaneously at 0 hr specimen. However, the cells labeled with both agents subsided when the cells were incubated in complete medium for 1 or 2 hr before fixation. Therefore, thymidylate synthase appears to exist mainly in G1-phase and to subside in early S-phase. Although the number of thymidylate synthase positive cells was greater than that of the cells labeled with tritiated thymidine, the ratio was constant (r = 0.99). The fraction of S-phase can be estimated from that of thymidylate synthase positive cells. Thymidylate synthase positive cell fraction may become another important segment for cell cycle analysis.