Iron-grown Acidithiobacillus ferrooxidans MON-1 cells are highly resistant to organomercurial compounds as well as mercuric chloride (HgCl2). Existence of a novel Hg2+-reducing enzyme system, in which mercury resistant aa3-type cytochrome c oxidase catalyzes the reduction of Hg2+ with reduced mammalian cytochrome c or Fe2+ as an electron donor to give Hg0, has been shown in iron-grown MON-1 cells. There has been no reports on the mechanism of Hg2+ reduction by sulfur-grown A. ferrooxidans cells. The level of mercury resistance in sulfur-grown A. ferrooxidans MON-1 cells was compared with that of iron-grown MON-1 cells. Strain MON-1 was able to grow in 1% elemental sulfur medium (pH 2.5) containing 10 μM of Hg2+ or 0.2 μM phenylmercury acetate (PMA), suggesting that the levels of mercury resistance to inorganic and organic mercurial compounds are nearly the same in iron- and sulfur-grown MON-1 cells. Activity levels of Hg0 volatilization from HgCl2, PMA, and methylmercury chloride (MMC) were also nearly the same in iron- and sulfur-grown cells and these activities were markedly activated by 100 mM of Fe2+, but strongly inhibited by 1 mM of sodium cyanide, indicating that sulfur-grown MON-1 cells has the activity of ferrous iron-dependent mercury reducing enzyme system containing aa3-type cytochrome oxidase. aa3-type cytochrome c oxidase purified partially from sulfur-grown MON-1 cells showed both the iron oxidase and mercury reductase activities in the presence, but not in the absence, of rusticyanin and c-type cytochromes (Cyc1 and Cyc2) partially purified from iron-grown MON-1 cells.