In previous studies, we have identified three promoters (P1, P2, and P3) in the regulatory region of the Escherichia coli aroP gene (P. Wang, J. Yang, and A. J. Pittard, J. Bacteriol. 179:4206-4212, 1997). Both P1 and P2 can direct mRNA synthesis for aroP expression, whereas P3 is a divergent promoter which overlaps with P1. The repression of transcription from the major promoter, P1, has been postulated to involve the activation of the divergent promoter, P3, by the TyrR protein (P. Wang, J. Yang, B. Lawley, and A. J. Pittard, J. Bacteriol. 179:4213-4218, 1997). In the present study, we confirmed the proposed mechanism of P3-mediated repression of P1 transcription by studying the binding of RNA polymerase to the promoters P1 and P3 in vitro in the presence and absence of TyrR protein and its cofactors. Our results show that (i) only one RNA polymerase molecule can bind to the DNA fragment carrying the aroP regulatory region, (ii) RNA polymerase has a higher affinity for P1 than for either P2 or P3 and binds to P1 in the absence of TyrR protein, (iii) in the presence of TyrR protein and its cofactor, phenylalanine or tyrosine, RNA polymerase preferentially binds to P3, and (iv) RNA polymerase does not respond to the activation-defective mutant TyrR protein TyrR-RQ10 and remains bound to P1 in the presence of TyrR-RQ10 and either of the cofactors.