Forward mutations induced by ethylmethane sulfonate (EMS) in the lacI gene of Escherichia coli were recovered from bacteria proficient or deficient in the alkyltransferase encoded by the constitutive ogt gene. EMS doses of 100 or 200 mM (Ogt+) and of 50 mM (Ogt−) were selected from the corresponding dose—response curves for DNA sequence analysis. A total of 239 induced mutations affecting the N-terminal region of the lacl gene were characterized. All mutations were G: C→A: T transitions, consistent with the predominant role of the O6-ethylguanine miscoding lesion in mutagenesis by EMS. In the Ogt+ spectrum at the lowest tested dose of 100 mM EMS, guanines preceded by an A or T base at the 5' side were on average 3.2 times more likely to mutate than those preceded by a G or C base. This bias diminished at the higher EMS dose (200 mM) and disappeared in the Ogt− genetic background. Previously reported data for Ogt+ bacteria in a Uvr-proficient background show an opposite bias in favor of mutations at guanines preceded by a G or C base. The overall 5' flanking base influence was estimated as 8-fold. These data suggest that DNA repair by Ogt alkyltransferase plays an important role in the processing of ethylation-induced lesions responsible for GC→AT transitions, influencing their ultimate distribution with respect to sequence context. The data further suggest that Ogt and UvrABC excision repair, the two major mechanisms of protection against the biological consequences of long-chain alkylating agents, show different DNA sequence specificity and that the relative importance of these two systems is highly dependent upon the chemical dose.