O6-carboxymethylguanine (O6-CMG) is a mutagenic DNA adduct that forms at increased levels when people eat meat. It has been studied as a potential initiating event in colorectal carcinogenesis. It can arise from alkylation of guanine in DNA by electrophilic degradation products of N-nitroso compounds. There is significant data regarding biochemical and cellular process, including DNA repair and translesion DNA synthesis that control O6-CMG accumulation, persistence, and mutagenicity. Mutation spectra arising from the adduct closely resemble common mutations in colorectal cancer; however, gaps remain in understanding the biochemical processes that regulate how and where the damage persists in the genome. Addressing such questions relies on advances in chemistry such as synthesis approaches and bioanalytical methods. Results of research in this area help advance our understanding of the toxicological relevance of O6-CMG-modified DNA. Further attention should focus on understanding how a combination of genetic and environmental factors control its biological persistence and how this information can be used as a basis of biomoniotoring and prevention efforts to help mitigate colon cancer risk.