The biosynthesis of 8-hydroxy-5-deazaflavin, a conspicuous cofactor in methane-producing bacteria, has been investigated in Methanococcus vannielii. Isotopic labeling studies with [U-14C]guanosine show that a form of guanine is used for biosynthesis of part of the deazaflavin molecule. This incorporation involves loss of the 8-carbon of the guanine moiety. Measurement of release of 14C from the 8-position of the [8-14C]guanine and [8-14C]GTP serves as the assay for the enzymic activity which carries out this reaction. The activity responsible for the reaction has been partially purified from crude extracts of M. vannielii.