A green fluorescent substance which was found in the culture filtrate of Clostridium acetobutylicum was found to be identical with “G Compound” extracted from the mycelium of Eremothecium ashbyii and was identified as 8-N-ribityl-6, 7-dimethyllumazine. This substance was observed to have a stimulative effect on the synthesis of riboflavin by growing and resting cells of the acetone-butanol fermentation bacterium. It was also found that this substance was converted enzymatically to riboflavin by the cell-free extract of Escherichia coli when incubated with a 4-C-donor. Of the compounds tested, pyruvate was most effective as a 4-C-donor of 8-N-ribityl-6, 7-dimethyllumazine in the riboflavin biosynthesis by both microorganisms.