The closely related Proteus mirabilis and Enterobacterlaceae plasmid-encoded urease genes are positively regulated by the AraC-like transcriptional activator UreR. In the presence of the effector molecule urea, UreR promotes transcription of ureD, the initial gene in the urease operon, and increases transcription of the divergently transcribed ureR. Here, we identify UreR-specific binding sites in the ureRp-ureDp intergenic regions. Recombinant UreR (rUreR) was expressed and purified, and gel shift and DNase I protection assays were performed with this protein. These analyses indicated that there are two distinct rUreR binding sites in both the plasmid-encoded and P. mirabilis ureRp-ureDp intergenic regions. A consensus binding site of TA/GT/CA/TT/GC/TTA/TT/AATTG was predicted from the DNase I protection assays. Although rUreR bound to the specific DNA binding site in both the presence and the absence of urea, the dissociation rate constant k-1 of the rUreR-DNA complex interaction was measurably different when urea was present. In the absence of urea, the dissociation of the protein-DNA complexes, for both ureRp and ureDp, was complete at the earliest time point, and it was not possible to determine a rate. In the presence of urea, dissociation was measurable with a k-1 for the rUreR-ureRp interaction of 1.2 +/- 0.2 x 10(-2) s-1 and a k-1 for the rUreR-ureDp interaction of 2.6 +/- 0.1 x 10(-3) s-1. This corresponds to a half-life of the ureRp-rUreR interaction of 58 s, and a half-life of the ureDp-rUreR interaction of 4 min 26 s. A model describing a potential role for urea in the activation of these promoters is proposed.