Assimilation of iron via aerobactin by Escherichia coli requires functions encoded by certain ColV plasmids and by the chromosomal fhu region. Mutants obtained by insertion of the transposons Mu d1 (Ap lac) and Tn5 into ColV-K311 revealed that the aerobactin genes are transcribed from the locus that determines aerobactin synthesis to the gene encoding the outer membrane receptor protein for ferric aerobactin uptake. The antibiotic streptonigrin was used for selection of mutants impaired in iron assimilation. A simple assay was devised to identify mutants devoid of aerobactin synthesis. In addition, we performed complementation studies between fhu mutants and fhu genes cloned into pBR322. The genes fhuC,D,B were required for uptake of ferric aerobactin. Mutations in the fhuA gene usually decreased expression of the subsequent fhuC,D,B genes to such an extent that growth on ferric aerobactin as sole iron source was abolished. The results suggest that the genes on the chromosome and on the ColV plasmid are organized in two operons.