The primary structure of the membrane bound segment of amphipathic bovine liver microsomal cytochrome b5 has been determined. This 43 residue nonpolar polypeptide is present at the COOH terminus of cytochrome b5. The sequence was obtained by automated sequence analysis and carboxypeptidase digestions. The sequence obtained is: Ile-Thr-Lys-Pro-Ser-Glu-Ser-Ile-Ile-Thr-Ile-Asp-Ser-Asn-Pro-Ser-Trp-Trp-Thr-Asn-Trp-Leu-Ile-Pro-Ala-Ile-Ser-Ala-Leu-Phe-Val-Ala-Leu-Ile-Tyr-His-Leu-Tyr-Thr-Ser-Glu-Asn. Conformational analysis using predictive algorithms is presented along with circular dichroism data on the peptide bound to phospholipid vesicles.