The genomes of Listeria spp. encode all but one of 25 enzymes required for the biosynthesis of adenosylcobalamin (AdoCbl; coenzyme B12). Notably, all Listeria genomes lack CobT, the nicotinamide mononucleotide:5,6- dimethylbenzimidazole (DMB) phosphoribosyltransferase (EC 188.8.131.52) enzyme that synthesizes the unique α-linked nucleotide N1-(5-phospho- α-d-ribosyl)-DMB (α-ribazole-5′-P, α-RP), a precursor of AdoCbl. We have uncovered a new pathway for the synthesis of α-RP in Listeria innocua that circumvents the lack of CobT. The cblT and cblS genes (locus tags lin1153 and lin1110) of L. innocua encode an α-ribazole (α-R) transporter and an α-R kinase respectively. Results from in vivo experiments indicate that L. innocua depends on CblT and CblS activities to salvage exogenous α-R, allowing conversion of the incomplete corrinoid cobinamide (Cbi) into AdoCbl. Expression of the L. innocua cblT and cblS genes restored AdoCbl synthesis from Cbi and α-R in a Salmonella enterica cobT strain. LinCblT transported α-R across the cell membrane, but not α-RP or DMB. UV-visible spectroscopy and mass spectrometry data identified α-RP as the product of the ATP-dependent α-R kinase activity of LinCblS. Bioinformatics analyses suggest that α-R salvaging occurs in important Gram-positive human pathogens. © 2010 Blackwell Publishing Ltd.