From crude protein extracts of Pseudomonas putida KT2440, we identified a small protein, TurA, able to bind to DNA fragments bearing the entire Pu promoter sequence of the TOL plasmid. The knock-out inactivation of the turA gene resulted in enhanced transcription initiation from the Pu promoter, initially suggesting a negative regulatory role of TurA on Pu expression. Ectopic expression of TurA both in P. putida and in Escherichia coli reporter strains and transcription in vitro of the Pu promoter in the presence of purified TurA confirmed the TurA repressor role on Pu activity. turA gene inactivation did not significantly alter two well characterized physiological regulations of the Pu expression in routine conditions of cultivation, exponential silencing, and carbon-mediated repression, respectively. However, the growth at suboptimal temperatures resulted in a TurA-dependent increase of Pu repression. These results strongly suggest that a physiological significance of the negative role of TurA on Pu activity could be limitation of the expression of the toluene-degrading enzymes at suboptimal growth temperatures. Therefore, the identification of TurA as Pu-binding protein revealed a novel physiological modulation of Pu promoter that is different from those strictly nutritional described previously.