Transposition activity in bacteria is generally maintained at a low level. The activity of mobile DNA elements can be controlled by bacterially encoded global regulators. Regulation of transposition of Tn4652 in Pseudomonas putida is one such example. Activation of transposition of Tn4652 in starving bacteria requires the stationary-phase sigma factor RpoS and integration host factor (IHF). IHF plays a dual role in Tn4652 translocation by activating transcription of the transposase gene tnpA of the transposon and facilitating TnpA binding to the inverted repeats of the transposon. Our previous results have indicated that besides IHF some other P. putida-encoded global regulator(s) might bind to the ends of Tn4652 and regulate transposition activity. In this study, employing a DNase I footprint assay we have identified a binding site of P. putida Fis (factor for inversion stimulation) centred 135 bp inside the left end of Tn4652. Our results of gel mobility shift and DNase I footprint studies revealed that Fis out-competes IHF from the left end of Tn4652, thereby abolishing the binding of TnpA. Thus, the results obtained in this study indicate that the transposition of Tn4652 is regulated by the cellular amount of P. putida global regulators Fis and IHF.