The cytotoxicity of SN1-type alkylating agents such as N-methyl-N′-nitrosourea (MNU), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), or the cancer chemotherapeutics temozolomide, dacarbazine and streptozotocin has been ascribed to the persistence of O6-methylguanine (meG) in genomic DNA. One hypothesis posits that meG toxicity is caused by futile attempts of the mismatch repair (MMR) system to process meG/C or meG/T mispairs arising during replication, while an alternative proposal suggests that the latter lesions activate DNA damage signaling, cell cycle arrest and apoptosis directly. Attempts to elucidate the molecular mechanism of meG-induced cell killing in vivo have been hampered by the fact that the above reagents induce several types of modifications in genomic DNA, which are processed by different repair pathways. In contrast, defined substrates studied in vitro did not undergo replication. We set out to re-examine this phenomenon in replication-competent Xenopus laevis egg extracts, using either phagemid substrates containing a single meG residue, or methylated sperm chromatin. Our findings provide further support for the futile cycling hypothesis.