Adrenal cytochrome b561 (cyt b561) is the prototypical member of an emerging family of proteins that are distributed widely in vertebrate, invertebrate and plant tissues. The adrenal cytochrome is an integral membrane protein with two b-type hemes and six predicted transmembrane helices. Adrenal cyt b561 is involved in catecholamine biosynthesis, shuttling reducing equivalents derived from ascorbate. We have developed an Escherichia coli system for expression, solubilization and purification of the adrenal cytochrome. The spectroscopic and redox properties of the purified recombinant protein expressed in this prokaryotic system confirm that the cytochrome retains a native, fully functional form over a wide pH range. Mass spectral analysis shows that the N-terminal signal peptide is intact. The new bacterial expression system for cyt b561 offers a sixfold improvement in yield and other substantial advantages over existing insect and yeast cell systems for producing the recombinant cytochrome for structure-function studies.