The biosynthesis of deazaflavins, flavins, ribonucleotides, and selected amino acids was studied in Methanobacterium thermoautotrophicum by incorporation of 13C-labeled acetate and pyruvate. 13C enrichments were monitored by 13C and 1H NMR spectroscopy. The biosynthesis of ribonucleotides follows the standard pathway. The xylene ring of riboflavin is formed from two pentose moieties in agreement with studies in yeasts and eubacteria. The pyrimidine ring and the ribityl side chain of the deazaflavin chromophore of coenzyme F420 are derived from the purine nucleotide pool. The phenolic ring and C-5 of the deazaflavin system are supplied by the shikimate pathway. A hypothetical mechanism for the assembly of the deazaflavin chromophore from 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione and 4-hydroxyphenyl-pyruvate is proposed.