The nucleotide sequence of the Escherichia coli fep genomic region has been determined. Three new loci were identified. One of these, P43, encodes a membrane protein that is not essential for ferric enterobactin transport. Two others, fepD and fepG, were found to be essential for transport and their translational products showed extensive homology to other integral membrane proteins involved in TonB-dependent transport processes. The FepC amino acid sequence suggested a peripheral membrane location and revealed conserved ATP-binding domains. Together these data indicate that ferric enterobactin is transported through a typical periplasmic binding protein-dependent system. In addition, the transcriptional organization of these genes was examined and primer extension analysis identified a single iron-regulated bidirectional promoter between the P43 gene and the fepDGC operon.