The iron-oxidizing system of a moderately thermophilic, extremely acidophilic, gram-positive mixotroph, Sulfobacillus sibiricus N1(T), was studied by spectroscopic, high-performance liquid chromatography and inhibitory analyses. Hemes B, A, and O were detected in membranes of S. sibiricus N1(T). It is proposed that the electron transport chain from Fe²(+) to O₂ is terminated by 2 physiological oxidases: aa₃-type cytochrome, which dominates in the early-exponential phase of growth, and bo₃-type cytochrome, whose role in iron oxidation becomes more prominent upon growth of the culture. Both oxidases were sensitive to cyanide and azide. Cytochrome aa₃ was more sensitive to cyanide and azide, with K(i) values of 4.1 and 2.5 µmol·L⁻¹, respectively, compared with K(i) values for cytochrome bo₃, which were 9.5 µmol·L⁻¹ for cyanide and 7.0 µmol·L⁻¹ for azide. This is the first evidence for the participation of a bo₃-type oxidase in ferrous iron oxidation. The respiratory chain of the mixotroph contains, in addition to the 2 terminal oxidases, a membrane-bound cytochrome b₅₇₃.