The CO----methylene blue and CO----dichlorophenol indophenol activities of carbon monoxide oxidase were specifically activated upon aerobic incubation with selenite, whereas the NADH----methylene blue activity was not altered. Fully active enzyme contained selenium, molybdenum, and flavin adenine dinucleotide in a 1:1:1 ratio. Selenium was covalently bound to the protein, probably between the sulfurs of half-cystine residues, and not a constituent of the molybdenum cofactor. The action of selenite was directed to the cytoplasmic species of carbon monoxide oxidase exclusively, whereas the CO----methylene blue activity of the membrane-bound enzyme remained unaffected.