<jats:p>A method was developed to study amino acids as possible precursors of PQQ biosynthesis. Cultures of <jats:italic>Hyphomicrobium</jats:italic> X, growing on [<jats:sup>13</jats:sup>C]methanol, were supplemented with unlabelled amino acids. Uptake and participation in metabolism were determined via gas chromatography/mass spectrometry of derivatized amino acids, obtained from hydrolysed cellular protein, by measuring their <jats:sup>12</jats:sup>C content. Several amino acids appeared to be incorporated into the protein to a significant extent, without degradation or conversion. Among these were the aromatic amino acids, L‐tyrosine and L‐phenylalanine. Using the same replacement approach, their incorporation into PQQ was determined by <jats:sup>1</jats:sup>H‐ and <jats:sup>13</jats:sup>C‐NMR spectroscopy of purified PQQ obtained from the culture medium. It appeared that the complete carbon skeleton of tyrosine was present, forming the <jats:italic>o</jats:italic>‐quinone and pyrrole‐2‐carboxylic acid moieties in PQQ, while phenylalanine was not incorporated at all. Starting with L‐tyrosine, possible biosynthetic routes to PQQ are discussed.