Six position-specific 13C-labelled isotopomers of glucose were supplied to the ectomycorrhizal fungi Suillus pungens and Tricholoma flavovirens. From the resulting distribution of 13C among fungal PLFAs, the overall order and contribution of each glucose atom to fatty acid 13C enrichment was: C6 (∼31%) > C5 (∼25%) > C1 (∼18%) > C2 (∼18%) > C3 (∼8%) > C4 (∼1%). These data were used to parameterize a metabolic model of the relative fluxes from glucose degradation to lipid synthesis. Our data revealed that a higher amount of carbon is directed to glycolysis than to the oxidative pentose phosphate pathway (60% and 40% respectively) and that a significant part flows through these pathways more than once (73%) due to the reversibility of some glycolysis reactions. Surprisingly, 95% of carbon cycled through glyoxylate prior to incorporation into lipids, possibly to consume the excess of acetyl-CoA produced during fatty acid turnover. Our approach provides a rigorous framework for analysing lipid biosynthesis in fungi. In addition, this approach could ultimately improve the interpretation of isotopic patterns at natural abundance in field studies.