Alkylating agents are S-phase-dependent clastogenic agents: Chromosome aberrations are not observed unless the treated cells have first undergone a replicative DNA synthesis. While DNA gaps resulting from misreplication of the alkylated template are believed to underlie aberration formation, the specific alkylated DNA lesions that produce these DNA gaps are not known. To quantitate the DNA strand break induction that results from replication of an alkylated DNA template and attempt to identify those alkylated lesions which underlie DNA strand breakage. [14C]thymidine-labeled Chinese hamster ovary (CHO) cells were treated with either N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or methyl methanesulfonate (MMS) in G1 and then allowed to progress through S phase in the presence of [3H]thymidine. When analyzed at the subsequent mitosis, DNA strand breaks were found in the nonalkylated ([3H]thymidine-labeled) DNA strand. This did not appear to be the consequence of any recombinational or endonuclease-mediated event and was more likely due to DNA gaps produced by incomplete replication off the alkylated template. A portion of these breaks probably result from a failure to replicate past 3-methyladenine. Differences between MNNG and MMS in the frequency of S-phase-dependent breaks they produce relative to the overall alkylation damage suggest that the O6-methylguanine lesion might also be involved in S-phase-dependent DNA strand breakage.