We have investigated hydroxyl free radical (.OH)-mediated damage to calf thymus DNA produced by potassium cyanide, kainate, H(2)O(2)/Fe(2+), or glutathione/Fe(3+) by in vitro method. When calf thymus DNA was exposed to potassium cyanide (0.5, 0.75, 1.0, 1.5, or 2.0 mM) or kainate (0.25, 0.5, or 1.0 mM) for 90 min at 37 degrees C in homogenate or the 9000g supernatant of mice brain, the quantity of DNA damage was observed to be concentration-dependent. Similarly, glutathione (1.0, 2.0, 4.0, 5.0, or 6.0 mM) inflicted damage on calf thymus DNA in the presence of Fe(3+) (3.0 microM). In addition, hydrogen peroxide (0.15, 0.30, 0.75, 1.50, or 3.0 mM) also caused damage to calf thymus DNA in the presence of Fe(2+) (3.0 microM) in the same manner. Furthermore, it was observed that the DNA damage induced by potassium cyanide (2.0 mM), kainate (0.5 mM), glutathione (4.0 mM)/Fe(3+), and H(2)O(2) (1.5 mM)/Fe(2+) was prevented by the treatment with melatonin (1.0 or 1.5 mM), a potent .OH scavenger. These results suggest that cyanide, kainate, glutathione/Fe(3+), and H(2)O(2)/Fe(2+)-mediated .OH may play a cardinal role for DNA damage induced by these chemicals. Hence the conclusion of the present study is that melatonin protects against DNA damage induced by the .OH produced by these chemicals.