Thetplgene ofCitrobacter freundiiencodes an enzyme that catalyzes the conversion ofl-tyrosine to phenol, pyruvate, and ammonia. This gene is known to be positively regulated by TyrR. The amplitude of regulation attributable to this transcription factor is at least 20-fold. Three TyrR binding sites, designated boxes A, B, and C, centered at coordinates −272.5, −158.5, and −49.5, respectively, were identified in the upstream region of thetplpromoter. The results of mutational experiments suggest that TyrR binds in cooperative fashion to these sites. The nonavailability of any TyrR site impairs transcription. Full TyrR-mediated activation oftplrequired integration host factor (IHF) and the cAMP receptor protein (CRP). By DNase I footprinting, it was shown that the IHF binding site is centered at coordinate −85 and that there are CRP binding sites centered at coordinates −220 and −250. Mutational alteration of the IHF binding site reduced the efficiency of thetplpromoter by at least eightfold. The proposed roles of CRP and IHF are to introduce bends intotplpromoter DNA between boxes A and B or B and C. Multimeric TyrR dimers were demonstrated by a chemical cross-linking method. The formation of hexameric TyrR increased whentplDNA was present. The participation of both IHF and CRP in the activation of thetplpromoter suggests that molecular mechanisms quite different from those that affect other TyrR-activated promoters apply to this system. A model wherein TyrR, IHF, and CRP collaborate to regulate the expression of thetplpromoter is presented.