The effect of paraquat or ultraviolet (UV) light exposure on calf thymus DNA was investigated in vitro. When paraquat (0.1, 0.25, 0.5, or 1.0 mM) was incubated with brain or lung homogenates prepared from mice in the presence of calf thymus DNA at 37°C for 90 min, paraquat inflicted damage to DNA in a concentration-dependent manner. However, DNA damage was completely abolished by co-treatment with melatonin (1.5 mM), a hydroxyl radical (⋅OH) scavenger. In addition, when paraquat (1.0 or 2.5 mM) was incubated with liver microsomes in the presence of calf thymus DNA and Fe3+ (3.0 μM) at 37°C for 90 min, DNA damage also occurred and was prevented by the co-treatment of melatonin (1.5 mM). DNA was also damaged by UV light exposure or when the Fenton reaction was induced; the Fenton reaction generates ⋅OH; again, the damage was blocked by the co-treatment of melatonin. These results suggest that ⋅OH induced by paraquat or UV light probably account for the DNA damage. In short, DNA damage induced by paraquat and UV radiation were completely prevented by co-treatment with the ⋅OH scavenger, melatonin.