Purpose: We investigated the radioprotective effect of endogenous melatonin release at different times associated with the circadian rhythm on head and neck radiotherapy. Materials and methods: Two groups of animals were subjected daily to 8 Gy single fraction radiotherapy in the head and neck region from 5:00 to 6:00 (the morning group) or from 19:00 to 20:00 (the evening group). Corresponding untreated groups served as controls. Submandibular glands from rats sacrificed on the seventh day after irradiation were assessed biochemically and histopathologically. Melatonin, malondialdehyde and superoxide dismutase levels in blood collected immediately prior to irradiation were measured with rat-specific ELISA kits. Results: In irradiated rats, melatonin, malondialdehyde and superoxide dismutase levels were significantly higher in the evening group than in the morning group. In nonirradiated rats, melatonin and superoxide dismutase levels were significantly higher in the evening group than in the morning group. The areas of seromucous acinar cells were similar between the irradiated and nonirradiated evening groups, but the area was higher in the evening irradiated group than in the morning irradiated group. Conclusion: Consideration of endogenous melatonin secretion associated with the circadian rhythm may offer new therapeutic solutions for the complications of head and neck radiotherapy.