Abstract Metallothionein (MT) has been shown to protect DNA against cleavage induced by a variety of mutagenic agents. The mechanism has been attributed to its ability to either chelate transitional metals that participate in the Fenton reaction, or scavenge free radicals by means of the abundant cystenyl residues of the proteins. In the present study, the protective action of MT against DNA cleavage by the copper–1,10-phenanthroline [(OP) 2Cu +] complex was studied in situ. At 0.1 μM, MT inhibited the (OP) 2Cu + induced DNA cleavage by about 50% (IC 50≈0.1 μM). At 2.5 μM, the cleavage activity was completely inhibited. Similar to MT, cysteine can protect against DNA cleavage by (OP) 2Cu + (IC 50 of approximately 3 mM), however, its action was 1500-fold less efficient than MT. The combined action of MT and cysteine was additive. Reduced glutathione (1 and 10 mM) did not protect the (OP) 2Cu + induced DNA cleavage. Sodium azide could inhibit the cleavage only at high concentrations (IC 40≈25 mM). Spectrophotometric analysis showed that MT can inhibit the formation of the DNA[(OP) 2Cu +] complex possibly by chelating Cu. It can also cause a dissociation of the complex after it was formed. In the later case, the mechanism through which MT protects against the DNA cleavage might occur when MT fitted in closely with the complex, competing with the hydroxyl groups of the nucleotides base for Cu, which, in turn, terminate the Fenton-like free radical reaction.