We have established a new purification procedure of cytochrome b 561 from bovine adrenomedullary chromaffin vesicles. The heme content analysis of the purified sample indicated the presence of 1.7 molecules of heme B/cytochrome b 561 molecule. EPR spectroscopy of the purified enzyme in oxidized state showed that there were three types of low spin heme species. Two of them showed usual EPR signals at g z ؍ 3.14 and g z ؍ 2.84 arising from the same heme and were interconvertible depending on pH. The other species showed a highly anisotropic low spin signal at g z ؍ 3.70, with a lower redox potential than the others, and a temperature-sensitive character. These properties are very similar to low potential cytochrome b (b L or b 566 ) of the mitochondrial complex III, indicating that the g z ؍ 3.70 species is derived from a heme component different from the one that shows the usual low spin EPR signals. Based on our new structural model, these two heme B prosthetic groups are likely to be located on both sides of the membranes in close contact with the ascorbic acid-and semidehydroascorbic acid-binding sites, respectively, to facilitate the electron transfer across the membranes. This molecular architecture may provide a structural basis for the transmembrane electron transfer catalyzed by this hemoprotein.