Ionizing radiation is a critical threat to biomolecules, especially DNA. Various combinatorial compounds have been studied to protect this biomolecule. Melatonin has been reported as a direct and indirect free radical scavenger, but in this study, we explored the effect of melatonin on assisting in DNA repair by expression of Cdkn1a and Rad50; both of these genes are involved in DNA repair signaling, induced by radiation in rat peripheral blood.Rats were irradiated with single whole-body linear accelerator X-ray radiation doses of 2 and 8 Gy with or without melatonin (100 mg/kg body weight) pretreatments. The rats were randomly divided into nine groups and given an intraperitoneal injection of melatonin or the same volume of vehicle alone 1 h before radiation. Blood samples were taken 8, 24, and 48 h postradiation to measure gene expression of Cdkn1a and Rad50 using quantitative reverse transcription polymerase chain reaction technique.Melatonin pretreatment increased the expression of Cdkn1a and Rad50 in 8 and 24 h postradiations (2 and 8 Gy) (P < 0.05), and there was no significant difference in 48 h postradiation compared to the radiation-only and vehicle plus radiation (2 and 8 Gy) groups.Based on our results, pretreatment with melatonin (100 mg/kg) may ameliorates injurious effects of 2 and 8 Gy ionization radiation by increasing the expression level of Cdkn1a and Rad50 in rat peripheral blood and assist in DNA double-strand breaks repair, especially during the early postradiation.