Plasmid pABN1 contains the genetic determinants for the aerobactin iron uptake system of plasmid ColV-K30. Transposon Tn1000 mutants of pABN1 defective in synthesis of a 50,000-dalton polypeptide were found neither to secrete nor to accumulate aerobactin, but were not impaired in iron transport functions, clearly indicating a role for this polypeptide in aerobactin biosynthesis. RNA-DNA hybridization studies with probes spanning the entire aerobactin gene cluster showed that the system is regulated at the transcriptional level by the availability of iron in the external medium. When induced by low-iron stress, all five genes of the cluster were transcribed at a uniformly high level. When repressed by excess iron, transcripts of the four biosynthesis genes were some 30-fold less abundant in the case of the parental ColV-K30 plasmid and 10-fold less for the recombinant plasmid pABN1, whereas the receptor gene in either plasmid was transcribed at only about a third of the induced level.