Mammalian cells have been classified as proficient (Mer(+)) or deficient (Mer(-)) in methyl excision repair in terms of their cytotoxic reactions to agents that form O(6)-alkylguanine and their abilities to reactivate alkylated adenoviruses. O(6)-Methylguanine (O(6)MeGua) is considered to be a lethal, mutagenic, and carcinogenic lesion. We measured the abilities of cell extracts to transfer the methyl group from an exogenous DNA containing O(6)MeGua to acceptor protein. The constitutive level of acceptor activity was independent of the Mer phenotype and was approximately 100,000 acceptor sites per cell. Treatment of cells with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) results in a dose-dependent decrease in the acceptor activity in extracts because the rapid reaction between endogenous O(6)MeGua and acceptor protein makes the latter unavailable for further reaction. Treatment of cells with 1 muM MNNG for 15 min or 2 muM for approximately 2 min uses up >95% of the constitutive activity. However, Mer(+) cells, which are resistant to MNNG, rapidly resynthesize new acceptor protein, and the activity returns to the basal level in approximately 90 min. In Mer(-) tumor cells and Chinese hamster cells, which are sensitive to MNNG, resynthesis is not detectable in 90 min. Mer(-) simian virus 40-transformed fibroblasts, known to have an intermediate sensitivity to MNNG, have an intermediate resynthesis rate. Treatment of cells with multiple low doses of MNNG results in the enhanced production of O(6)MeGua-accepting protein in levels 2.5-fold above the constitutive values for Mer(+) tumor cells and to approximately 1.5-fold for Mer(+) fibroblasts or Mer(-) simian virus 40-transformed cells. Such treatments reduce the activities in Mer(-) tumor cells and Chinese hamster cells. We conclude: (i) estimates of O(6)MeGua in cellular DNA shortly after treatment may be seriously in error because of the rapid repair of this lesion, and (ii) the adaptive resynthesis of acceptor protein, not its constitutive level, is the important correlate of cell resistance to methylating agents.