In anaerobic bacteria, glycine, formate, and the amide-N of glutamine are building blocks for the biosynthesis of the imidazole moiety of the vitamin B12-base 5,6-dimethylbenzimidazole. These building blocks are also used for the biosynthesis of the imidazole moiety of purine bases. Therefore we tested 4(5)-aminoimidazole, the base moiety of the purine nucleotide precursor 5-aminoimidazole ribonucleotide, for its putative function as precursor of 5,6-dimethylbenzimidazole. The anaerobic vitamin B12-producer Eubacterium limosum, grown in the presence of [2-13C]4(5)-aminoimidazole, synthesized nonlabeled vitamin B12, but also [2-13C]7-azabenzimidazolylcobamide and [2-13C]5,6-dimethyl-7-azabenzimidazolylcobamide. [2-13C]limidazole was used by E. limosum to form [2-13C]imidazolylcobamide. Simultaneously nonlabeled vitamin B12 was synthesized. This shows that 4(5)-aminoimidazole and imidazole are not intermediates in the biosynthesis of 5,6-dimethylbenzimidazole. However, 4(5)-aminoimidazole has obviously a structure similar to the structure of an as yet unknown precursor of the vitamin B12-base, and is therefore transformed into the aza analogs. In order to prepare a reference compound 4(5)-azabenzimidazole was added to a culture of Propionibacterium shermanii and to a culture of E. limosum, P. shermanil transformed this base mainly to 4-azabenzimidazolylcobamide, as determined by 1H NMR-spectroscopy (NOE experiment). In contrast E. limosum produced mainly 7-azabenzimidazolylcobamide. The reason for this difference is discussed.