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A site-specifically modified oligonucleotide containing a single 2′-deoxyribonolactone lesion was used as a template for primer extension reac-tions catalyzed by M-MuLV reverse transcriptase (RT) and by the Klenow fragments of Escherichia coli DNA polymerase proficient (KF exo+) or deficient (KF exo–) in exonuclease activity. Analysis of the extension products in the presence of the four dNTPs or of a single dNTP showed that the M-MuLV RT was completely blocked and did not incorporate any dNMP opposite 2′-deoxyribonolactone. KF exo – pref-erentially incorporated nucleotides opposite the lesion following the frequency order dAMP> dGMP>> dTMP ~ dCMP and thus appeared to obey the ‘A rule ’ for preferential incorporation as has been shown previously for the 2′-deoxyribose abasic site. In the sequence context examined, the primer exten-sion by KF exo – appeared to be less efficient when dAMP was positioned opposite the lesion as compared with dTMP or dGMP. These two nucleo-tides promoted a more efficient polymerization accompanied by nucleotide deletion through misalignment incorporations. We therefore predict that the sequence context may strongly influence the translesional synthesis by KF exo – and thus the miscoding and mutational potential of the 2′-deoxyri-bonolactone in E.coli