The amino acid sequence of the heme-binding domains of rat liver cytochromes b5 from outer mitochondrial membranes and from microsomes has been determined by a combination of automatic and manual degradation of fragments generated by trypsin digestion and by cleavage at tryptophan. Tryptic peptides were separated by high-pressure liquid chromatography. The sequence of microsomal cytochrome b5 is identical with the one published by Ozols and Heinemann after completion of this study [Biochim. Biophys. Acta (1982) 704, 163-173]. The sequence of outer membrane cytochrome b5 differs from the microsomal one at 38 positions out of 91. There are 40 positions invariant between this sequence and the eight microsomal sequences published thus far. The non-conservative substitutions are located at the surface of the known three-dimensional structure of calf microsomal cytochrome b5 except for the substitution of histidine-15 by arginine. This paper brings the final proof that two iso-cytochromes b5 exist in the same cell. Their high degree of similarity as well as their differential cellular localization raise some questions which are briefly discussed.