In this study we addressed the question as to whether the mutagenesis by methylating agents is affected by the transcriptional activity of the damaged gene. An Epstein-Barr virus (EBV)-derived shuttle vector system was developed where the genetic target for mutation analysis, the bacterial gpt gene, is under the control of an eukaryotic inducible promoter in plasmid pF1-EBV and lacks the eukaryotic promoter in plasmid pF2-EBV. Two human cell lines that episomically maintain these shuttle vectors were established. In clone 6NT cells, which contain pF1-EBV plasmid, the gpt gene is actively transcribed and the transcription rate is regulated by zinc ions. In clone 3 cells, which harbor pF2-EBV plasmid, the gpt gene is not transcribed. Following treatment of both cell lines with the potent alkylating carcinogen N-methyl-N-nitrosourea (MNU), G.C to A.T transitions were the major mutagenic event, consistent with the miscoding potential of O6-methylguanine. The mutations were predominantly generated in the non-transcribed DNA strand of the active gpt gene. The same strand-bias was observed when the gpt gene was transcriptionally inactive, indicating that MNU-induced strand-specific formation of mutations is not due to transcription. Our data identify as major determinants of this phenomenon the sequence-specificity of MNU mutagenesis and the conformational properties of the target protein. Differences in mutation distribution were observed between the transcriptionally active and inactive gpt gene. This finding suggests that the organization of active genes in chromatin might modulate DNA alkylation and/or DNA repair.