The pCF10 plasmid in Enterococcus faecalis transfers from donor cells to recipients upon induction via peptide pheromone. Two plasmid-encoded negative regulators produced from the same transcript, PrgX protein and Qa RNA, repress conjugation genes in uninduced donor cells. PrgX positively autoregulates production of both itself and mature Qa RNA, and is believed to repress the prgQ promoter in a pheromone-sensitive fashion. Previous analysis of PrgX was complicated because mutations in prgX affecting regulation of conjugation also disrupted PrgX autoregulation, suggesting the two functions might be inseparable. In this study, we isolated 14 single amino acid substitutions in PrgX that reduced or eliminated repression of prgQ, without affecting autoregulation or DNA binding. PrgX was shown to bind to its cognate pheromone, cCF10, and most of the mutations lowered the affinity of PrgX for cCF10. Dimerization was affected by five of the mutations and the data indicate that it is required, but insufficient for pheromone induction. We propose a new model for the mechanism used by PrgX for regulation of the prgQ promoter, PrgX autoregulation, and Qa RNA processing.