The cytochrome b(5) tail is a 43-residue membrane-embedded domain that is responsible for anchoring the catalytic domain of cytochrome b(5) to the endoplasmic reticulum membrane. Different models for the structure of the membrane domain of cytochrome b(5) have been proposed, including a helical hairpin and a single transmembrane helix. In the present study, CD spectroscopy was used to investigate the conformation of the tail in different environments, and as a function of temperature, with the goal of understanding the nature of the membrane-bound conformation. Whereas residue property profiling indicates that bending of a helix in the middle of the peptide might be possible, the experimental results in small unilamellar vesicles and lysophosphatidylcholine micelles are more consistent with a single transmembrane helix. Furthermore, although there is evidence for some refolding of the polypeptide with temperature, this is not consistent with a hairpin-to-transmembrane transition. Rather, it appears to represent an increase in helical content in fluid lipid environments, perhaps involving residues at the ends of the transmembrane segment.