The Enterococcus faecalis conjugative plasmid pPD1 encodes proteins responsible for the mating response to the sex pheromone cPD1 secreted by a recipient cell. This response involves the respectively negative and positive determinants traA and traE, the pheromone-inhibitor determinant ipd and structural genes participating in the conjugation process. TraA is capable of binding to key sites within the regulatory gene cluster. The binding of TraA to cognate sites is modulated by the pheromone (cPD1) and the pheromone-inhibitor (iPD1) peptides. Using atomic force microscopy and classic biochemical techniques, we mapped and characterized the TraA-DNA interactions within the pPD1 regulatory gene cluster and the role of TraA in the transcription regulation of the sex pheromone response. A previous report showed that TraA binds to three adjacent sites (tab1, tab2 and tab3) located upstream of the ipd promoter region. Here, we provide direct evidence for such interactions and show that TraA alone or in the presence of iPD1 inhibits ipd transcription by preferentially binding to tab1, whereas in the presence of saturating cPD1, the overall binding to the tab sites decreases, TraA preferentially binds to tab3 and the ipd repression is relieved. Moreover, TraA alone or in the presence of iPD1 binds to two non-adjacent sites within the ipd terminators T1 and T2, an interaction that is also relieved in the presence of cPD1. The binding of TraA to the termination region of ipd may play an important role in controlling traE and traF expression via a transcriptional read-through mechanism already postulated for the pAD1 plasmid. TraA may also regulate its own expression by binding to a site in the proximity of the traA promoter, which has been relocated 200 bp downstream of the ipd gene. A model for the TraA-mediated regulation of the pPD1-encoded sex pheromone response is presented.