The promoters of the mar/sox/rob regulon of Escherichia coli contain a binding site (marbox) for the homologous transcriptional activators MarA, SoxS and Rob. In spite of data from footprinting studies, the marbox has not been precisely defined because of its degeneracy and asymmetry and seemingly variable location with respect to the -10 and -35 hexamers for RNA polymerase (RNP) binding. Here, we use DNA retardation studies and hybrid promoters to identify optimally binding 20 bp minimal marboxes from a number of promoters. This has yielded a more defined marbox consensus sequence (AYnGCACnnWnnRYYAAAYn) and has led to the demonstration that some marboxes are inverted relative to others. Using transcriptional fusions to lacZ, we have found that only one marbox orientation is functional at a given location. Moreover, the functional orientation is determined by marbox location: marboxes that are 15 or more basepairs upstream of the -35 hexamer are oriented opposite those closer to the -35 hexamer. Marbox orientation and the spacing between marbox and signals for RNP binding are critical for transcriptional activation, presumably to align MarA with RNP.