[1-13C]Glycine, [2-13C]glycine, and [15N]glycine were added to fermentations with the strictly anaerobic vitamin B12 producer Eubacterium limosum. The vitamin B12 isolated was almost exclusively labeled in its 5,6-dimethylbenzimidazole moiety. It was methylated and degraded to 1,5,6-trimethylbenzimidazole. The position of the 13C label was determined from the 13 C NMR spectrum using the natural-abundance 13C NMR spectrum of 1,5,6-trimethylbenzimidazole as reference. Thus it was found that C-1 of glycine is incorporated into position 7a, C-2 of glycine into position 3a of the 5,6-dimethylbenzimidazole moiety of vitamin B12. In order to show whether the nitrogen atom of glycine is also incorporated, the vitamin B12 from the experiment with [15N]glycine was degraded to 5,6-dimethylbenzimidazole. The 1H NMR spectrum of this compound exhibited in the region of the C-2 proton a doublet from the 15N-labeled part of the molecules (35%) and a singlet from the unlabeled molecules. This doublet indicates that only one of the two nitrogen atoms of 5,6-dimethylbenzimidazole is labeled. From the incorporation pattern of 13C-labeled glycine it must be deduced that this is N-3 of the 5,6-dimethylbenzimidazole moiety of vitamin B12. These experiments reveal that one molecule of glycine is incorporated into 5,6-dimethylbenzimidazole moiety of vitamin B12 in a regiospecific manner.