Thiobacillus ferrooxidans AP19-3 oxidized molybdenum blue (Mo5+) enzymatically. Molybdenum oxidase in the plasma membrane of this bacterium was purified ca. 77-fold compared with molybdenum oxidase in cell extract. A purified molybdenum oxidase showed characteristic absorption maxima due to reduced-type cytochrome oxidase at 438 and 595 nm but did not show absorption peaks specific for c-type cytochrome. The optimum pH of molybdenum oxidase was 5.5. The activity of molybdenum oxidase was completely inhibited by sodium cyanide (5 mM) or carbon monoxide, and an oxidized type of cytochrome oxidase in a purified molybdenum oxidase was reduced by molybdenum blue, indicating that cytochrome oxidase in the enzyme plays a crucial role in molybdenum blue oxidation.