The extent of degradation of the fungal biomass in forest soil during laboratory incubation was investigated as a measure of ectomycorrhizal (EM) biomass. The method simulates the disappearance of fungal mycelium after root trenching, where the EM fungi, deprived of its energy source (the tree), will start to die off. Incubating a forest humus soil at 25 C resulted in a decrease in the relative proportion (mol%) of the phospholipid fatty acid 18:2omega6,9 (a fungal marker molecule) within 3-6 months, indicating that fungal biomass was disappearing. Incubation at 5 degreesC resulted in essentially no change in the amount of 18:2omega6,9. The measurement of ergosterol, another fungal marker molecule, gave similar results. Incubation of different forest soils (pine, spruce and spruce/oak), and assuming that the disappearance of fungal biomass during this period of time was entirely due to EM fungi, resulted in an estimation of EM biomass of between 47 and 84% of the total fungal biomass in these soils. The humus layer had more EM biomass than deeper mineral layers.