The incorporation of 13C- and 14C-labeled precursors into 5-deaza-7,8-didemethyl-8-hydroxyriboflavin (factor F0) was studied with growing cells of Methanobacterium thermoautotrophicum. 5-Amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione was incorporated into the deazaflavin and into riboflavin without dilution. Tyrosine and 4-hydroxyphenylpyruvate were incorporated into the deazaflavin and into cellular protein. 4-Hydroxybenzaldehyde was not incorporated. A reaction mechanism is proposed for the formation of the deazaflavin chromophore from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and tyrosine or 4-hydroxyphenylpyruvate.