The molybdenum cofactor of formylmethanofuran dehydrogenase from methanol-grown Methanosarcina barkeri was isolated as the [difcarboxami-domethyl)]-derivative. The alkylated factor showed an absorption spectrum and chemical properties identical to those recently reported for the molybdenum cofactor of dimethyl sulfoxide reductase from Rhodobacter sphaeroides. By treatment with nucleotide pyrophosphatase the factor was resolved into two components, which were identified as [di(carboxamidomethyl)]-molybdopterin and GMP by their absorption spectra, their retention times on Lichrospher RP-18, and by their conversion to dephospho-[di(carboxamidomcthyl)]-molybdopterin and guanosine. respectively, in the presence of alkaline phosphatase. The GMP-moiety was sensitive to pcriodate, identifying it as the 5'-isomer. These results demonstrate that the molybdenum cofactor isolated from formylmethanofuran dehydrogenase contains the phosphoric anhydride of molybdopterin and 5'-GMP.