The integration host factor (IHF) of Pseudomonas putida connects cell growth to transcriptional activity of distinct promoters. The IHF site of the σ54 promoter Pu of the TOL (m-xylene biodegradation) plasmid pWW0 of P. putida has been examined to define experimentally a relationship between occupation of the promoter by this factor, the biological activity of the protein and the tolerance of the target site to single-base changes through the bound DNA core sequence. The use of an in vivo high-intensity UV imprinting procedure to examine such an occupation of Pu by IHF allowed inspection of the interplay between the factor and cognate site variants under the physiologically relevant conditions of monocopy gene dosage. The resulting data were merged in a structural model for establishing key features of the IHF–DNA interaction. A functional consensus for first-order IHF binding was instrumental for a genome-wide survey of sequences with potential regulatory value. This search revealed that very few, if any, of the maximum 330 sites within intergenic regions were placed in locations controlling expression of central metabolic genes. It thus seems that the IHF regulon of P. putida has a degree of functional specialization that is not evenly distributed through all gene categories.