The tpl gene of Citrobacter freundii encodes an enzyme that catalyzes the conversion of L-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 the tpl promoter. 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 of tpl required 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 the tpl promoter by at least eightfold. The proposed roles of CRP and IHF are to introduce bends into tpl promoter 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 when tpl DNA was present. The participation of both IHF and CRP in the activation of the tpl promoter 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 the tpl promoter is presented.