Riboflavin, the biosynthetic precursor of the 5,6-dimethylbenzimidazole moiety of vitamin B 12 , is transformed non-enzymatically into 5,6-dimethylbenzimidazole in small yield on treatment with 1 ɴ or 5 ɴ NaOH at 100 °C. Besides 5,6-dimethylbenzimidazole 1,2-diamino-4,5-dimethylbenzene, 1.2- dihydro-6,7-dimethyl-2-keto-1-ᴅ-ribityl-3-quinoxaline carboxylic acid and N-1-ᴅ-ribitylamino-2- amino-4,5-dimethylbenzene can be detected. When [1′- 14 C] riboflavin is used the 5,6-dimethylbenzimidazole contains about 75 per cent of the specific radioactivity of riboflavin. N-1-ᴅ-ribityl- amino-2-amino-4,5-dimethylbenzene is transformed into 5,6-dimethylbenzimidazole more efficiently than riboflavin. Oxygen enhances the yield of 5,6-dimethylbenzimidazole and 1,2-diamino-4,5-di- methylbenzene from riboflavin as well as from N-1-ᴅ-ribitylamino-2-amino-4,5-dimethylbenzene. 1.2- diamino-4,5-dimethylbenzene reacts together with formaldehyde but not with formate to form 5,6-dimethylbenzimidazole under alkaline conditions at 100 °C. It is therefore suggested that the nonenzymatic reaction of riboflavin proceeds via N-1-ᴅ-ribityl- amino-2-amino-4,5-dimethylbenzene and 1,2-diamino-4,5-dimethylbenzene, and that the latter reacts with formaldehyde preferably formed by oxidative degradation of C-1′ of the ribityl side chain to form 5,6-dimethylbenzimidazole via its unstable imidazoline derivative. The possible relevance of these results for the enzymatic process is discussed.