Molybdenum cofactor (mocofactor) is extracted efficiently, free of impurities and in high concentrations, by acid treatment of xanthine oxidase and subsequent incubation of the precipitate with phosphate buffer containing EDTA, molybdate and oxygen. It is suggested that cofactor is bound to the enzyme via hydrophobic forces as well as via an oxygen-sensitive mechanism. Upon extraction, the capability to complement the apo nitrate reductase of Neurospora crassa nit-1 can be conserved only in the total absence of oxygen. Cysteine and glutathione were shown to protect efficiently free mocofactor from oxidation. Two species of active mocofactor, probably a molybdoform and a demolybdoform, could be separated by means of reversed-phase HPLC with a mobile phase of 5 mM sodium citrate at a pH of 6.5. The mode of interaction between either of these species with thiol reagents is discussed.