Cobamides such as vitamin B12 (cobalamin) are produced exclusively by prokaryotes and used by many other organisms as cofactors for diverse metabolic processes. Cobamides are cobalt-containing tetrapyrroles with upper and lower axial ligands. The structure of the lower ligand varies in cobamides produced by different bacteria. We investigated the biochemical basis of this structural variability by exploring the reactivity of homologs of CobT, the enzyme responsible for activating lower ligand bases for incorporation into cobamides. Our results show that CobT enzymes can activate a range of lower ligand substrates, and the majority of the enzymes tested preferentially attach 5,6-dimethylbenzimidazole (DMB), the lower ligand of cobalamin. This suggests that many bacteria that synthesize cobamides other than cobalamin in pure culture may produce cobalamin in mixed communities by attaching DMB when it is available in the environment.