Methanoculleus thermophilicum was shown to contain two pterin derivatives. The structures of these pterin derivatives were established from amino acid analysis, 1H-NMR and fast-atom bombardment mass spectrometry data. One of the pterins was identified as tatiopterin-O, an aspartyl derivative of methanopterin with a proton at position 7 of the pterin moiety. The other pterin, which we named thermopterin, differed in the structure of the aniline group, containing two additional hydroxyl residues. The IUPAC name of thermopterin is N-[-1'-(2"-amino-4"-hydroxy-6"-pteridinyl)ethyl]-4- [2',3',4',5'-tetrahydroxypent-1'-yl(5'----1") O-alpha-ribofuranosyl-5"-phosphoric acid]-2,5-dihydroxyaniline, in which the phosphate group is esterified with alpha-hydroxyglutarylaspartic acid.