The genetic determinants for the aerobactin iron uptake system of plasmid ColV-K30, cloned as recombinant plasmid pABN1, were mapped by insertional inactivation using Tn1000 (gamma delta). Sites of insertion resulting in loss of aerobactin biosynthesis spanned ca. 5.5 kilobase pairs of cloned ColV-K30 DNA contiguous with a 2-kilobase-pair region in which transposon insertion resulted in loss of the outer membrane ferric-aerobactin receptor protein. Translation products of plasmid pABN1, and of subclones specifying siderophore biosynthesis alone or receptor activity alone, were analyzed by using the maxicell and minicell expression system. Four polypeptides (Mr = 62,000, 35,000, 45,000, and 50,000) are required for biosynthesis of aerobactin. A fifth product (Mr = 74,000) of plasmid pABN1 represents the outer membrane receptor protein. The linear order of genes for these polypeptides was determined by comparing translation products of a series of smaller derivative plasmids and of a number of mutant plasmids carrying Tn1000 at known locations.