The rhl quorum sensing (QS) circuit of Pseudomonas aeruginosa is known to regulate the expression of a number of virulence factors. This study investigates the regulation of rhlI, encoding the auto-inducer synthase RhlI responsible for the synthesis of N-butryl-L-homoserine lactone (BHL). A putative RpoN binding site was located upstream, in the promoter region of rhlI. Utilising a rhlI-lacZ transcriptional reporter, we demonstrate that under certain media conditions RpoN is a positive regulator of rhlI transcription. Measurements of BHL in extracted supernatant showed that the transcriptional patterns were reflected in the BHL levels, which were reduced in the rpoN mutant. Elastase and pyocyanin, known to be regulated by the rhl QS circuit, were shown to be reduced in a RpoN deficient strain. However, exogenous addition of BHL to the rpoN mutant did not restore these phenotypes suggesting that other regulatory factors apart from BHL are involved. Consistent with other rhl regulated phenotypes, we found that a rpoN mutant strain forms a biofilm that is different from that of the wild-type but similar to that displayed by a rhlI mutant.