ERV9 is a low repeated family of human endogenous retroviral elements whose expression is mainly detectable in undifferentiated embryonal carcinoma NT2/D1 cells. To define all the elements required for the correct transcription activity of the ERV9 promoter and to establish a precise correlation between the elements important for basal transcription, we have systematically analyzed the in vivo and in vitro transcriptional activity of many different ERV9 promoter mutants, including a series of linker-scanning mutations across the promoter region. We report here that the ERV9 promoter contains two elements controlling the selection of the correct start sites, a TATA box and an Inr-like region; the concerted action of both elements is necessary for faithful transcription. Finally, using a series of GAL4 protein fusion constructs in cotransfection experiments, we demonstrated that various transcription factors can synergistically induce a high level of transcription when bound to an ERV9 DNA promoter.