Cell extracts prepared from cells of Methanosarcina barkeri grown on hydrogen and carbon dioxide, acetate, or methanol contain a coenzyme structurally related to methanofuran. This modified coenzyme was highly purified and its structure assigned as 4-[N-(gamma-L-glutamyl-gamma-L-glutamyl-gamma-L-glutamyl-gamma-L-glutamy l)-p- (beta-amino-ethyl)phenoxymethyl]-2-(aminomethyl)furan. The key structural evidence was obtained by high-resolution fast atom bombardment-mass spectrometry and 1H NMR spectroscopy. Quantitative analysis of the hydrolytic fragments of the coenzyme supported the assigned structure. We propose that this coenzyme be called methanofuran-b.