The DNA adduct O(6)-methylguanine (O(6)MeG) induced by environmental genotoxins and anticancer drugs is a highly mutagenic, genotoxic and apoptotic lesion. Apoptosis induced by O(6)MeG requires mismatch repair (MMR) and proliferation. Models of O(6)MeG-triggered cell death postulate that O(6)MeG/T mispairs activate MMR giving rise to either direct genotoxic signaling or secondary lesions that trigger apoptotic signaling in the 2(nd) replication cycle. To test these hypotheses, we used a highly synchronized cell system competent and deficient for the repair of O(6)MeG adducts, which were induced by the S(N)1 methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We show that DNA double-strand breaks (DSBs) are formed in response to O(6)MeG at high level in the 2nd S/G(2)-phase of the cell cycle. This is accompanied by ATR and Chk1 phosphorylation, G(2)/M arrest and late caspase activation. Although cells undergo apoptosis out of the 2nd G(2)/M-phase, the majority of them recovers and undergoes apoptosis after passing through additional replication cycles. The late apoptotic effects were completely abolished by O(6)-methylguanine-DNA methyltransferase, indicating that non-repaired O(6)MeG is carried over into subsequent generations, eliciting there a late apoptotic response. We also demonstrate that with a low, non-toxic dose of MNNG the passage of cells through the 1st and 2nd S-phase is not delayed, although the dose is able to induce excessive sister chromatid exchanges. This suggests that a significant amount of O(6)MeG can be tolerated by recombination, which is a fast and highly efficient process preventing from S-phase blockage, DSB formation and cell death.