The prgB gene encodes aggregation substance (Asc10) which is essential for transfer of the pheromone-inducible conjugative plasmid pCF10 in Enterococcus faecalis. The prgQ and prgS regions, located 4 kb upstream of prgB, are required for the expression of prgB. Complementation studies indicated that the prgQ region functions in cis and in an orientation-dependent manner relative to the prgB gene (J. W. Chung and G. M. Dunny, Proc. Natl. Acad. Sci. USA 89:9020-9024, 1992). Analysis of transcriptional fusions in this study, using a promoterless lacZ gene in several locations between prgQ and prgB, confirmed that the prgQ region does not carry a promoter for the expression of prgB and that prgB does not comprise an operon with prgA (which encodes the surface exclusion protein Sec10), the gene immediately upstream from prgB. Northern (RNA) blot analysis demonstrated that two distinct transcripts (Q(S) RNA and Q(L) RNA), much larger than the prgQ gene, were expressed in the prgQ region. Q(S) RNA was produced constitutively, whereas Q(L) RNA was produced inducibly by pheromone. The lack of any other open reading frame in Q(L) RNA and significant sequence complementarity between the 3' end of Q(L) RNA and the promoter region of prgB suggested that the functional products of the prgQ region might be RNA molecules rather than proteins. A mutation in prgS completely abolished the production of Q(L) RNA. A model for transcriptional activation of prgB is presented.