The corrinoid cofactor of the tetrachloroethene reductive dehalogenase of Dehalospirillum multivorans was isolated in its Coβ-cyano form. This cofactor represents the main corrinoid found in D. multivorans cells. Analysis of the isolated cyano-corrinoid by a combination of HPLC and UV/VIS-absorbance spectroscopy revealed it to be nonidentical to a variety of known natural B12 derivatives. From high-resolution mass-spectrometric analysis, the molecular formula of the corrinoid isolated from D. multivorans could be deduced as C58H81CoN17O14P. The sample of the novel corrinoid from D. multivorans was further analyzed by UV/VIS, CD, and one- and two-dimensional 1H-, 13C-, and 15N-NMR spectroscopy, which indicated its structure to be closely related to that of pseudovitamin B12 (Coβ-cyano-7″-adeninylcobamide). By the same means, the corrinoid could be shown to differ from pseudovitamin B12 only by the lack of the methyl group attached to carbon 176, and, therefore, it was named norpseudovitamin B12 (or, more precisely, 176-norpseudovitamin B12). Norpseudovitamin B12 represents the first example of a ‘complete’ B12-cofactor that lacks one of the methyl groups of the cobamide moiety, indicating that the B12-biosynthetic pathway in D. multivorans differs from that of other organisms. X-Ray crystal-structures were determined for norpseudovitamin B12 from D. multivorans and the analogues pseudovitamin B12 and factor A (Coβ-cyano-7″-[2-methyl]adeninylcobamide). These first accurate crystal structures of complete corrinoids with an adeninyl pseudonucleotide confirmed the expected coordination properties around Co and corroborated the close conformational similarity of the nucleotide moieties of norpseudovitamin B12 and its two homologues.