The low-potential electron carrier, coenzyme F420, was purified from Methanobacterium strain M.o.H. A yield of 160 mg/kg of wet-packed cells was obtained. Results of analysis of hydrolytic fragments and periodate oxidation products of the coenzyme, by infrared, UV-visible, 1H and 13CNMR spectrometry, mass spectrometry, and quantitative elemental analyses indicate that coenzyme F420 is: N-[N-[O-[5-(8-hydroxy-5-deazaisoalloxazin-10-yl)-2,3,4-trihydroxy-4-pentoxyhydroxyphosphinyl]-L-lactyl]-gamma-L-glutamyl]-L-glutamic acid. A convenient trivial name would be the N-(N-L-lactyl-gamma-L-glutamyl)-L-glutamic acid phosphodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin 5'-phosphate. Proof of structure by organic synthesis was not performed; the stereochemical configuration of the hydroxyl groups on the side chain as well as the position of the hydroxyl group on the aromatic ring require confirmation by organic synthesis of the molecule.