The genes fhuA, fhuC and fhuD encode products involved in the transport of ferrichrome into cells of Escherichia coli. The nucleotide sequence of an additional locus contiguous to fhuD and formerly designated fhuB was determined. It contains an open reading frame for a polypeptide that consists of 659 amino acids. Expression of plasmids containing the fhuB region in an in vitro transcription/translation system and in E. coli minicells revealed a polypeptide with an electrophoretic mobility on SDS-polyacrylamide gels extremely dependent on the experimental conditions employed. This property could be explained by the very hydrophobic nature of the protein and it was concluded that the fhuB locus consists of a structural gene that encodes a membrane protein with a molecular weight of 82,181. The fhuB gene is required for all iron transport systems which operate via hydroxamate compounds. The order of the genes in the fhu operon is fhuA fhuC fhuD fhuB, and transcription proceeds from fhuA to fhuB.