In vivo and in vitro analyses indicate that transcription of the argO gene coding for an arginine exporter is regulated by the global transcriptional regulator Lrp, an effect that went by unnoticed in previous genome-scale screenings of the Lrp regulatory network in Escherichia coli. Lrp activates the argO promoter fourfold; exogenous leucine antagonizes, but does not completely eliminate this effect. Activation by Lrp interferes with the previously demonstrated activation of the argO promoter by ArgP. This interference results from the mutual inhibitory binding of the two activators to overlapping targets. As a consequence, each regulator acts more potently in the absence of the other. Dimeric Lrp binds cooperatively to at least three regularly spaced semi-palindromic binding sites. Leucine reduces complex formation approximately twofold but concomitantly enhances the cooperativity of the binding. Footprinting data suggest a severe Lrp-induced deformation of the argO control region. Combined, the effector modulated activation of argO transcription by ArgP and Lrp must ensure an adapted and fine-tuned synthesis of the transporter in response to environmental conditions. The repertoire of bacterial transcription regulation mechanisms is vast, but the competitive activation of a single promoter by two activator proteins as described here appears to be rare.