Two kinds of high molecular mass cytochrome c3 (Hmc) were isolated from Desulfovibrio vulgaris Miyazaki, one from the membrane (mHmc), the other from the soluble fraction (sHmc). Molecular masses of both Hmcs determined by SDS-PAGE are 65 kDa. Each Hmc molecule is composed of a single polypeptide chain and contains 16 hemes. sHmc and mHmc have identical UV-visible absorption spectra of a typical c-type cytochrome (three peaks at 530, 419, and 355 nm in the ferri form, and four peaks at 553, 523, 419, and 325 nm in the ferro form), and similar amino acid composition. There is no peak at 695 nm in the ferri form, but a Soret peak (423 nm) with a shoulder at 432 nm, indicative of the presence of a high-spin heme, appeared at low reduction stage. Hmcs are fragile proteins and readily denatured by bubbling with gas or by stirring. From the redox titration of sHmc monitored spectroscopically in the presence of standard redox dyes, the midpoint potentials of 16 hemes (E1 approximately E16) were determined. These hemes are classified into four groups based on their Eis: positive (E1 = 60, E2 = 15 mV), slightly negative (E3 = E4 = E5 = E6 = E7 = -120, E8 = -125, E9 = -135 mV), negative (E10 = E11 = -190, E12 = E13 = -195, E14 = -205 mV), and very negative (E15 = E16 = -260 mV) groups (may be deviated from the true microscopic values due to heme-heme interactions). The role of Hmc in the energy metabolism of this bacterium is discussed.