Previous work has demonstrated that fleR, the gene for a transcriptional activator belonging to the NtrC subfamily of response regulators, is involved in the regulation of mucin adhesion and flagellar expression by Pseudomonas aeruginosa. This report describes the identification and characterization of fleQ, the gene for another transcriptional regulator which also regulates mucin adhesion and motility in this organism. The complete nucleotide sequence of the fleQ gene was determined on both DNA strands, and an open reading frame (ORF) consisting of 1,493 nucleotides was identified. This ORF coded for a gene product of predicted molecular weight, as confirmed by the overexpression of the fleQ gene as a fusion protein under an inducible promoter. The fleQ gene is flanked by a flagellar operon, fliDSorf126, at the 5' end and the fleSR operon on the 3' end. FleQ also had striking homology to a number of proteins belonging to the NtrC subfamily of response regulators, which work in concert with the alternate sigma factor RpoN (sigma54) to activate transcription. However, FleQ lacks the residues corresponding to Asp-54 and Lys-104 of the NtrC protein which are conserved in most of the members belonging to this subfamily of regulators. In addition, unlike some of the other transcriptional activators of this group, FleQ does not appear to have a cognate sensor kinase. A chromosomal insertional mutation in the fleQ gene abolished mucin adhesion and motility of P. aeruginosa PAK and PAK-NP. Both of these functions were regained by providing the complete fleQ gene on a multicopy plasmid. The location of fleQ immediately upstream of the fleSR operon, which is also necessary for the same process, suggested that these regulators may interact in some way. We therefore examined the regulation of the fleSR operon by fleQ and vice versa. Promoter fusion experiments showed that the fleSR operon was regulated by RpoN and FleQ. On the other hand, the fleQ promoter was independent of RpoN and FleR. FleQ, thus, adds another level of regulation to motility and adhesion in P. aeruginosa, above that of fleSR. We therefore propose the existence of a regulatory cascade which consists of at least two transcriptional regulators, FleQ and FleR, in the control of motility and adhesion in P. aeruginosa.