Expression of conjugative transfer and virulence functions of the Enterococcus faecalis antibiotic resistance plasmid pCF10 is regulated by the interaction of the pheromone receptor protein PrgX with two DNA binding operator sites (XBS1 and XBS2) upstream from the transcription start site of the prgQ operon (encoding the pCF10 transfer machinery) and by posttranscriptional mechanisms. Occupancy of both binding sites by PrgX dimers results in repression of the prgQ promoter. Structural and genetic studies suggest that the peptide pheromone cCF10 functions by binding to PrgX and altering its oligomerization state, resulting in reduced occupancy of XBSs and increased prgQ transcription. The DNA binding activity of PrgX has additional indirect regulatory effects on prgQ transcript levels related to the position of the convergently transcribed prgX operon. This has complicated interpretation of previous analyses of the control of prgQ expression by PrgX. We report here the results of in vivo and in vitro experiments examining the direct effects of PrgX on transcription from the prgQ promoter, as well as quantitative correlation between the concentrations of XBSs, PrgX protein, and prgQ promoter activity in vivo. The results of electrophoretic mobility shift assays and quantitative analysis of prgQ transcription in vitro and in vivo support the predicted roles of the PrgX DNA binding sites in prgQ transcription regulation. The results also suggest the existence of other factors that impede PrgX repression or enhance its antagonism by cCF10 in vivo.