The fepA gene of Escherichia coli encodes the outer-membrane receptor protein for ferrienterobactin. Previous genetic studies indicated that fepA mutations occur frequently and suggested that most of the mutations were deletions. In this work seven spontaneous fepA mutations were analyzed by enzyme assay (enterobactin synthase and enterobactin esterase) and by DNA hybridization studies. In two strains, UT500 and UT700, the mutations were confined to the fepA gene. In the remaining mutants, the mutations were large deletions; in several cases, 27 kb or more of DNA had been lost. The deletions, all of which eliminated approximately the left half of the enterobactin gene cluster, extended from the vicinity of the fepC gene counterclockwise into the chromosome. A minimum of three clockwise endpoints were identified and at least two counterclockwise endpoints were detected. The variation in endpoints among the deletions argues against the involvement of a normal transposon in their formation. Also, unexpected homology was found between enterobactin gene cluster DNA and lacPOZ and pSC101.