Histone deacetylase (HDAC) inhibitors are undergoing clinical evaluation for cancer therapy. Because HDAC modulates chromatin structure and gene expression, parameters considered to influence radioresponse, we have investigated the effects of the HDAC inhibitor MS-275 on the radiosensitivity of two human tumor cell lines (DU145 prostate carcinoma and U251 glioma). Acetylation status of histones H3 and H4 was determined as a function of time after MS-275 addition to and removal from culture medium. Histone acetylation increased by 6 h after MS-275 addition, reaching a maximum between 24 and 48 h of exposure; providing fresh drug-free medium then resulted in a decrease in histone acetylation that began by 6 h and approached untreated levels by 16 h. Treatment of cells with MS-275 for 48 h followed by irradiation had little or no effect on radiation-induced cell death. However, exposure to MS-275 before and after irradiation resulted in an increase in radiosensitivity with dose enhancement factors of 1.9 and 1.3 for DU145 and U251 cells, respectively. This MS-275 treatment protocol did not result in a redistribution of the cells into a more radiosensitive phase of the cell cycle or in an increase in apoptosis. However, MS-275 did modify the time course of γH2AX expression in irradiated cells. Whereas there was no significant difference in radiation-induced γH2AX foci at 6 h, the number of cells expressing γH2AX foci was significantly greater in the MS-275-treated cells at 24 h after irradiation. These results indicate that MS-275 can enhance radiosensitivity and suggest that this effect may involve an inhibition of DNA repair.