Purpose: To investigate the effect of the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (DAC), on radiosensitivity in breast cancer cells. Materials and methods: Two breast cancer cell lines, MDA-MB-231 and MDA-MB-435, were evaluated. The methylation status and the mRNA expression of three genes (ER, PR, and HIC-1) that were frequently hypermethylated in these cell lines were determined as a function of DAC exposure. 3-(4,5)-dimethylthiahiazo-(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) absorbance and a colony-forming assay were used to estimate cell viability and radiosensitivity. Using cell-cycle analysis, gamma-histone H2A (gamma-H2AX) formation assays and methylation-specific polymerase chain reaction (MSP) analysis of three genes correlated with radiosensitivity (BRCA1, 14-3-3 sigma, and E-cadherin), the mechanism of DAC enhancement of radiosensitivity was explored. Results: DAC induced the demethylation and reactivation of silent genes in both cell lines. The combination of DAC and irradiation induced growth suppression in vitro. DAC, 5 mu M, enhanced sensitivity to ionizing radiation. DAC followed by irradiation correlated with G2/M arrest and the retardation of repair of radiation-induced double strand breaks. DAC reversed the methylation status of genes connected with radiosensitization. The different radiosensitization effects observed with different breast cancers cells may correlate with the primary methylation status of radiosensitizing genes. Conclusions: Treatment strategies that include DAC present promising options for sensitizing breast cancer cells to irradiation.