The gene of mammalian endonuclease III homologs (NTHL1/Nthl1), a DNA glycosylase/AP lyase involved in base excision repair, lies immediately adjacent to one of the tuberous sclerosis disease-determining genes, TSC2/Tsc2, in a head-to-head orientation. To clarify the regulation of these divergent genes, we studied the promoter activities of these transcription units by luciferase assay using HeLa cells. We found that the short spacer sequence of 63 base pairs (bp) between the mouse Nthl1 and Tsc2 genes shows bidirectional promoter activity essential for the transcription of both genes, The 63-bp sequence is well conserved among several mammalian species and contains two Ets-transcription factor binding sites (EBSs) in opposite directions. An Ets-family protein in the HeLa nuclear extract specifically bound to either EBSs. Mutation of the core motif of the EBS demonstrated that EBS positively regulates transcription of both mNthl1 and mTsc2 genes, These EBSs had an additive effect on transcription, and each EBS functioned equally in both directions.