Objective: to evaluate the protective effect of melatonin on peripheral blood mononuclear cells (PBMCs) exposed in vitro to ionizing radiation. Materials and methods: PBMCs drawn from healthy volunteers were incubated with 0, 1x10-5, 1x10-6 y 1x10-7 M melatonin for 10 minutes before being exposed to gamma radiation (300 cGy; Co60 source). Afterwards, DNA damage was evaluated with the comet assay. Results: PBMCs pretreated with melatonin showed comet tails shorter than those without the hormone treatment, as well as a lower percentage of cells with severe DNA damage. Conclusion: melatonin doses of 1x10-5, 1x10-6 and 1x10-7 M provide in vitro protection to PBMCs from DNA damage (single strand breaks and alkali-labile sites) induced by gamma radiation (300 cGy; Co60 source).