Aerobactin and enterobactin, hydroxamate- and catechol-type siderophores, respectively, were found capable of removing Fe (III) from transferrin in buffered solution. Although under these conditions aerobactin displaced the Fe much more slowly than did enterobactin, the rate for the former could be accelerated by addition of PPi as mediator. Transfer to Fe(III) from transferrin to aerobactin appeared to proceed via a ternary complex. Cells of Escherichia coli BN 3040 NalR iuc containing transport systems for both enterobactin and aerobactin, the genetic determinants for the latter specified on a ColV-type plasmid, took up Fe from [55Fe]transferrin in minimal medium. In this case aerobactin was effective at a much lower concentration, although enterobactin still displayed superior ability to transfer the Fe. In serum, however, the rate measured with aerobactin exceeded that found with enterobactin. Aerobactin, in spite of its relatively unimpressive affinity for Fe(III) as a siderophore, is nonetheless equipped with structural features or properties that enhance its ability to remove the metal ion from transferrin, especially when receptor-bearing cells of E. coli are present to act as a thermodynamic sink for the Fe. These attributes of the aerobactin system of Fe assimilation may account for its status as a virulence determinant in hospital isolates of E. coli.