The conversion of dihydroneopterin triphosphate in the presence of 6-pyruvoyl tetrahydropterin synthase was followed by 1H-NMR spectroscopy. The interpretation of the spectra of the product is unequivocal: they show formation of a tetrahydropterin system carrying a stereospecifically oriented substituent at the asymmetric C(6) atom. The spectra are compatible with formation of a (3')-CH3 function, and with complete removal of the 1' and 2' hydrogens of dihydroneopterin triphosphate. The fast-atom-bombardment/mass spectrometry study of the same product yields a [M + H]+ ion at m/z 238 compatible with the structure of 6-pyruvoyl tetrahydropterin. The data support the proposed structure of 6-pyruvoyl tetrahydropterin as a key intermediate in the biosynthesis of tetrahydrobiopterin.