The complete nucleotide sequence of the Escherichia coli cybB gene for diheme cytochrome b561 and its flanking region was determined. The cybB gene comprises 525 nucleotides and encodes a 175 amino acid polypeptide with a molecular weight of 20,160. From its deduced amino acid sequence, cytochrome b561 is predicted to be very hydrophobic (polarity 33.7%) and to have three membrane spanning regions. Histidines, canonical ligand residues for protohemes, are localized in these regions, and the heme pockets are thought to be in the cytoplasmic membrane. No significant homology of the primary structure of cytochrome b561 with those of other bacterial b-type cytochromes was observed.