Certain ColV plasmids of Escherichia coli contain genes that specify functions for the acquisition of iron(III) via aerobactin. The locus for aerobactin synthesis of pColV-K311 was cloned into pBR322. Mutagenesis with the transposon Tn1000, and the generation of deletions with restriction enzymes resulted in multicopy plasmids which complemented pColV mutants impaired in various steps of aerobactin synthesis. The insertion and deletion mutants were mapped and assigned to three loci termed aerA, aerB, and aerC. It is proposed that these genes mediate the synthesis of aerobactin by specifying functions for hydroxylation (aerA), acetylation of the 6-amino group of 6-hydroxylysine (aerB), and the coupling of N-acetyl-N-hydroxy-lysine with citrate (aerC). The order and transcription polarity of the structural genes was found to be aerB, aerC, aerA.