Quantitative cleavage of peptide bonds adjacent to tryptophanyl and methionyl residues in the polar moiety of cytochrome b5 was effected using cyanogen bromide in the presence of heptafluorobutyric and formic acids. Application of this method to native cytochrome b5 resulted in cleavage at tryptophanyl and methionyl residues in the polar and membranous segments in high yield. Amino acid analysis of peptides isolated from such digests indicated that tyrosine was modified and the derivative eluted in a position preceding lysine; however, the color constant with ninhydrin remained unchanged. Hydriodic acid hydrolysis of the phenylthiohydantoin derivative of the modified tyrosine regenerated the parent amino acid. Peptides containing the altered tyrosine remained susceptible to chymotryptic cleavage at this residue. Cleavage of methionyl bonds could be prevented by methylene blue-sensitized photooxidation prior to cyanogen bromide/anhydrous heptafluorobutyric acid treatment. The conditions employed for photooxidation were selective for methionyl residues, and the yield and specificity of tryptophanyl cleavage by the cyanogen bromide/anhydrous heptafluorobutyric acid method was unaffected.