The pCF10-encoded negative regulators PrgX and Qa (prgQ antisense) RNA inhibit pCF10 transfer by blocking prgQ transcription extension past a potential transcription terminator sequence IRS1. To identify potential target sites for negative regulation, we isolated and analysed 13 cis-acting mutations in the prgXQ region. Determination of the 3′ end of Qa RNA showed that eight mutations mapped in the region encoding Qa RNA. Four mutations were in the Qa promoter region and one was in IRS1. Three mutations in Qa greatly reduced the intracellular level of this RNA but did not affect that of PrgX. However, both Qa RNA and PrgX protein were reduced in three Qa promoter region mutants and the expression of prgQ transcripts extending 3′ from IRS1 became constitutive. Qa RNA could mediate its negative regulatory activity in the absence of PrgX, and this activity was not abolished by cCF10, the peptide pheromone that induces pCF10 transfer. RNA analysis showed that Qa RNA abolished transcription readthrough. Based on the experimental data as well as computer analysis of predicted secondary structures of prgQ mRNA in the presence or absence of Qa, we concluded that Qa RNA is a pheromone-insensitive effector of prgQ mRNA termination or degradation at IRS1. In cells lacking a Qa target sequence, expression of PrgX repressed transcription from the prgQ promoter, and this repression was relieved by addition of exogenous cCF10. Thus, even though the synthesis of these negative regulators is coupled, they each act independently on separate targets to regulate expression of conjugation functions.