Growth of A. ferrooxidans ATCC 23270 cells in sulfur medium with 0.005% ferric sulfate for 3, 4, 5, 6, 7 and 10 days gave the maximum growth yield of 45, 58, 76, 86, 90 and 95 mg protein per liter medium, respectively. Iron oxidase activities of 1-, 2- and 3- day-cultured cells on sulfur with 0.005% ferric sulfate (3.4, 3.5 and 0.8 μmol Fe2+ oxidized/mg protein/min) were approximately 68, 70 and 16% of iron-grown ATCC 23270 cells (5.0 μmol/mg protein/min). In contrast iron oxidase activities of 1-, 2- and 3-day cultured cells on sulfur without iron (4.9, 3.8 and 2.7 μmol Fe2+ oxidized/mg protein/min) were approximately 98, 76 and 54% of the iron oxidase activity observed in iron-grown ATCC 23270 cell. SFORase activities of 3 day-cultured cell on sulfur with and without ferric sulfate (0.62 and 0.31 μmol Fe2+ produced/mg protein/min) were approximately 20 and 10 fold higher than that of iron-grown cell (0.03 μmol Fe2+ produced/mg protein/min). Both iron oxidase and SFORase activities increased at early-log phase and decreased at late-lag phase during growth of the strain on sulfur with or without Fe3+. The plasma membranes which had iron oxidase activity were prepared not only from iron-grown cells but also sulfur-grown cells. Iron oxidase activities of the plasma membranes prepared from sulfur- and iron-grown cells were 3.6 and 4.5 nmol Fe2+ oxidized per mg protein per min. These results suggest that iron oxidation enzyme system has a role in part in the energy generation of this bacterium from sulfur.