Secretion of aerobactin is thought to play an important part in the virulence of invasive Escherichia coli also capable of synthesizing enterochelin. Why, despite its markedly lower affinity for iron than that of enterochelin, aerobactin proves to be the predominant active siderophore for bacterial growth in transferrin was investigated. We studied the action of two iron chelators, 2,2'-dipyridyl and transferrin, in expression of the aerobactin and enterochelin genes. Specifically, we describe the sequential localization of the two siderophores in the cell compartments during bacterial growth under different iron limitation conditions. Our results demonstrated that, whatever the exogenous iron-chelating agent used, aerobactin was rapidly excreted, whereas enterochelin accumulated early in periplasm before its very belated release into the external medium. This work also showed that the advantage of aerobactin over enterochelin in competition with transferrin was not due to (i) lack of enterochelin activity, (ii) a cell-bound aerobactin-dependent mechanism, (iii) antagonism between the two siderophores, and probably (iv) genetic preferential induction of aerobactin. We propose that the superiority of aerobactin in competing with transferrin for iron(III) was a consequence of its more rapid excretion with respect to enterochelin. In contrast to transferrin, 2,2'-dipyridyl induced a greater efficiency of enterochelin, possibly by a more permanent function as iron-binding compounds in the bacterial envelope. In summary, unlike aerobactin, enterochelin appears to be a weakly secreted high-affinity iron ligand.