The complete amino acid sequence of rat cytochrome b5 has been determined. Isolation of this species of cytochrome b5 in its native form from microsomes by means of detergent solubilization required the inclusion of the protease inhibitor, phenylmethylsulfonyl fluoride, throughout the isolation steps. Omission of the protease inhibitor yielded a 97-residue heme-containing peptide without the membranous segment. The primary structure of the intact molecules was deduced from automated sequence analysis of peptides generated by proteolytic or chemical cleavage and isolated exclusively by reversed-phase HPLC. The blocked amino terminus of cytochrome b5 was identified as N-acetylalanine. The hexosamine content of the cytochrome preparation was less than 0.1 mol/mol protein, indicating an absence of asparaginyl linked oligosaccharide.