PrgX negatively regulates prgQ transcriptional read-through in the pheromone-inducible enterococcal conjugative plasmid pCF10. We isolated and characterized 13 dominant-negative prgX mutants, all of which mapped in either the N- or the C-terminus of PrgX. In all mutants, the in vivo level of Qa RNA, an antisense RNA to prgQ RNA, was greatly reduced. When oligomerization of PrgX was tested with a phage lambda cl repressor fusion system, the oligomerization domain was found to be between amino acid residues 78 and 280. When histidine-tagged PrgX (His-PrgX) was purified by nickel column chromatography from a strain also expressing PrgX, PrgX was co-purified with His-PrgX. Although PrgX was expressed at a much higher level than His-PrgX, an approximately equal amount of PrgX was co-purified. Pheromone induction greatly decreased the co-purification of PrgX. Based on these data, we propose that both the N- and the C-terminal domains of PrgX are required for PrgX positive autoregulation and for the repression of prgQ transcription read-through. In vivo, PrgX exists as a dimer, and dimerization is mediated by the central region of PrgX.