Resonance energy transfer was used to study the structure of cytochrome b5 and its nonpolar segment reconstituted into sonicated vesicles of dimyristoylphosphatidylcholine. The n-(9-anthroyloxy) (AO) fatty acid probes were added to these vesicles, and energy-transfer measurements were carried out between tryptophan and AO, tryptophan and the heme moiety of cytochrome b5, and AO and heme. Results of these measurements were analyzed by using the methods outlined in the previous paper [Kleinfeld, A. M. (1985) Biochemistry (preceding paper in this issue)]. We find, in agreement with Fleming et al. [Fleming, P. J., Koppel, D. E., Lau, A. L. Y., & Strittmatter, P. (1979) Biochemistry 18, 5458-5464], that the fluorescent tryptophan in both forms of the protein is buried about 20 A from the surface and that most of the fluorescence is associated with a single tryptophan. The results are consistent with the AO probe distance of closest approach to the protein, greater for whole b5 than for the nonpolar peptide. The tryptophan-heme and AO-heme measurements indicate that the heme moiety is about 15 A from the surface of the membrane. The agreement of our results with the previous studies supports the description of tryptophan-AO energy transfer outlined in the preceding paper.