The amphipathic flavoprotein NADH-cytochrome b5 reductase from steer liver was converted into the membrane-binding and soluble catalytic domains by controlled subtilisin proteolysis of vesicle-bound, externally oriented reductase. Amino acid analysis of the single nonpolar peptide which remained associated with the vesicles showed that it has a molecular weight of about 6400-6500 and that 65-66% of the amino acid residues are hydrophobic. Both the intact reductase and the nonpolar peptide were blocked to sequencing. Carboxypeptidase Y digestion of the holoenzyme and of a chymotryptically generated flavopeptide, which had lost membrane-binding capacity, released 1 residue of alanine and 2 residues of phenylalanine, indicating a common carboxyl terminus. Partial sequence analysis of the reductase placed the nonpolar peptide to the NH2-terminal side of the subtilisin-generated flavopeptide domain. Thus, the membrane-binding domain of cytochrome b5 reductase is localized at the NH2-terminus of the whole protein. The nonpolar peptide bound to vesicles contains a single fluorescent tryptophan with an emission maximum characteristic of a hydrophobic environment.