The molybdenum cofactor isolated from sulfite oxidase (sulfite: ferricytochrome c oxidoreductase, EC 1.8.2.1) and xanthine dehydrogenase (xanthine:NAD+ oxidoreductase, EC 1.2.1.37) in the presence of iodine and KI (form A) has been shown to contain a pterin nucleus with an unidentified substituent in the 6 position [Johnson, J. L., Hainline, B. E. & Rajagopalan, K. V. (1980) J. Biol. Chem. 255, 1783-1786]. A second inactive form of the cofactor was isolated aerobically but in the absence of iodine and KI. The latter cofactor derivative (form B) is highly fluorescent, has a visible absorption band at 395 nm and, like form A, contains a phosphate group. Cleavage of the phosphate ester bond with alkaline phosphatase exposes a glycol function that is sensitive to periodate. Oxidation of form B with alkaline permanganate yields a highly polar compound with properties of a sulfonic acid, suggesting that the active molybdenum cofactor might contain sulfur. The sulfur-containing pterin urothione characterized by Goto et al. [Goto, M., Sakurai, A., Ohta, K. & Yamakami, H. (1969) J. Biochem. 65, 611-620] had been isolated from human urine. The permanganate oxidation product of urothione, characterized by Goto et al. as pterin-6-carboxylic-7-sulfonic acid, is identical to that obtained from form B. Because urothione also contains a periodate-sensitive glycol substituent, a structural relationship is suggested. The finding that urine samples from patients deficient in the molybdenum cofactor are devoid of urothione demonstrates a metabolic link between the two molecules.