The members of the histone deacetylase (HDAC) family play important roles in various cellular processes, including transcriptional regulation, cell proliferation, differentiation and apoptosis. Inhibitors of histone deacetylases are emerging as an important new class of chemotherapeutic agents. As such, identifying stable and potent chemical HDAC inhibitory compounds is an important focus for translational research. Here we report the results of a rational drug design of novel HDAC inhibitors with potential for sensitizing cancer cells to radiation therapy. Over 60 HDAC inhibitor analogues incorporating a urea backbone and the hydroxamic acid end moiety were designed and screened. Six were found to confer 50% inhibition of HDAC enzyme activity at nanomolar concentrations. These candidate HDAC inhibitors inhibited cell proliferation at the ranges of IC50 10-50 microM in various cancer cells, including prostate (PC-3), breast (MCF-7) and head and neck squamous carcinoma (SQ-20B). Furthermore, radiation clonogenic survival assays revealed that these compounds possess radiosensitizing properties that are cell type-specific. The data support the further investigation of these HDAC inhibitors for use as sensitizing agents with potential for clinical application.