Abstract Glioblastoma is one of the most aggressive tumor in the central nervous system tumors, with 5-year survival rates of less than 10%. The standard therapy for glioblastomas is maximal safe resection, followed by radiation therapy and chemotherapy with temozolomide (TMZ). One of the reasons of the worse prognosis of the tumor is the acquisition of resistance to TMZ in glioma cells. TMZ is a DNA-methylating agent. TMZ-induced O6-methylguanine adducts, in the absence of repair by O6-methylguanine DNA methyltransferase (MGMT), mispair with thymine during the next DNA replication cycle, leading to futile DNA mismatch repair (MMR) by the MMR system, formation of double strand breaks (DSBs) and eventual cell death of glioma cells. Several mechanisms have been proposed about the acquisition of resistance to TMZ in glioblastomas, such as changes of expression of MGMT, MMR, nucleotide excision repair and homologous recombination (HR). To clarify the mechanisms of resistance to TMZ and to find the way to overcome the resistance to TMZ, several clones of U251 cells resistant to TMZ were obtained and analyzed. FACS analyses showed several patterns of cell cycle distributions of the cells treated with TMZ. #3 resistant clone showed G2 arrest at day3 after TMZ exposure and this arrest was abrogated sooner compared to the G2 arrest of parental U251. TMZ did not induce G2 arrest in #8 resistant clone. Consistent with the results, TMZ-induced DSBs in #3 resistant cells were rapidly disappeared compared to those in U251 cells, and the DSBs were least induced by TMZ in #8 resistant cells. The expression of MGMT was not found in U251 parental cells, #3 cells nor #8 cells, which mean MGMT was not associated with the resistance to TMZ in those cells. The protein levels of MSH6, which was associated with MMR, was reduced in #8 cells. The inhibitor of PLK1 or PARP reduced the cell growth independent of MMR. The ability of HR was increased in #3 resistant clone. By suppression of Rad51, which was major factor associated with HR, the resistant #3 clone was re-sensitized to TMZ, however #8 was not. These results suggested that inhibition of HR may be a viable means to resensitize some kinds of temozolomide-resistant gliomas to TMZ. Citation Format: Shigeo Ohba, Yuichi Hirose. The mechanisms of resistance to temozolomide in glioma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2845.