AbstractKluyveromyces marxianus has a high potential for industrial production of aroma compounds, such as 2‐phenylethanol, which is derived in a bioconversion from L‐phenylalanine. In the present work the product yield of K. marxianus in batch cultivation was estimated as 0.65 mol 2‐phenylethanol/mol L‐phenylalanine and thus significantly below the theoretical optimum of 1 mol/mol. By a comprehensive approach of stoichiometric balancing and GC–MS analysis of various substrates and products of K. marxianus a detailed insight into its metabolism was gained. For this purpose ring‐labelled (13C6) L‐phenylalanine and naturally labelled glucose were applied as substrates in tracer studies in batch culture. The produced aroma compounds 2‐phenylethanol and 2‐phenylethylacetate stem exclusively from the supplied L‐phenylalanine, whereas glucose was not converted into these products because of efficient feed‐back inhibition of prephenate dehydratase in the L‐phenylalanine biosynthetic pathway. It could be further shown that the supplied L‐phenylalanine completely covers the anabolic cellular demand for this amino acid. Quantification of 13CO2 in the exhaust gas provided clear evidence for catabolic breakdown of L‐phenylalanine during cultivation. Metabolic balancing around the pool of free intracellular L‐phenylalanine revealed a significant loss of L‐phenylalanine into catabolic and anabolic pathways. While 73.3% of L‐phenylalanine was converted into 2‐phenylethanol or 2‐phenylethylacetate, 22.4% was catabolized through the cinnamate pathway and 4.3% was directed towards protein biosynthesis. Catabolic breakdown of L‐phenylalanine via hydroxylation to L‐tyrosine could be excluded. In addition to an insight into metabolic functioning and regulation of 2‐phenylethanol‐producing K. marxianus, the approach presented here provides important information on potential targets for genetic optimization of 2‐phenylethanol‐producing yeasts. Copyright © 2002 John Wiley & Sons, Ltd.