The primary structure of the membranous segment of porcine liver microsomal cytochrome b5 has been determined. This polypeptide is at the COOH terminus of the cytochrome molecule and consists of 43 amino acids. It is essential for the insertion of the cytochrome into the endoplasmic reticular membrane. Automated sequence analysis of tryptic and cyanogen bromide/anhydrous heptafluorobutyric acid peptides provided data from which the following unique amino acid sequence was deduced: Ile-Ala-Lys-Pro-Ser-Glu-Thr-Leu-Ile-Thr-Thr-Val-Glu-Ser-Asn-Ser-Ser-Trp-Trp-Thr-Asn-Trp-Val-Ile-Pro-Ala-Ile-Ser-Ala-Leu-Val-Val-Ser-Leu-Met-Tyr-His-Phe-Tyr-Thr-Ser-Glu-Asn. A prediction of alpha-helices, beta-structures, and beta-turns basedon the sequence of this polypeptide is also presented.