Bifunctional chloroethylating cytostatic agents produce lethal DNA lesions, as a result of the formation of O6-alkylguanines. These lesions can be repaired by O6-methylguanine-DNA methyltransferase (MGMT). This ubiquitous nuclear and cytosolic enzyme removes the alkyl group by accepting it to the cysteine residue of its active site, thus preventing the formation of DNA interstrand cross-links. The role of the circadian organization in cellular protection against such DNA insults was examined in male B6D2F1 mice, synchronized with an alternation of 12 h of light and 12 h of darkness (LD12:12). MGMT activity was determined in liver of mice obtained at eight different circadian times, located 3 h apart. MGMT activity varied 5-fold along the 24 h time-scale, from 7 +/- 1 pmol/g of tissue at 7 h after light onset (HALO), during the rest span, up to 32 +/- 9 pmol/g at 19 HALO (second mid to late activity span). This large amplitude circadian rhythm in MGMT activity may be an important determinant of the susceptibility rhythms to alkylating agents. The greatest DNA repair activity occurred at night when mice were active, eat and drink, and thus are at a higher risk of being exposed to chemical insults.