The composition of fatty acids and sterols in soil lipid fractions is often used as a global indicator for the status and changes of soil microbial communities. In order to validate such analyses in the context of ectomycorrhizal communities, an experiment was performed in which seedlings of Pinus sylvestris and the fungus Pisolithus tinctorius were grown separately, or combined to form ectomycorrhiza under axenic conditions. Fatty acids of the neutral lipid fraction (NLFAs) and the phospholipid fraction (PLFAs) as well as sterols were identified and quantified by gas chromatography–mass spectrometry. When grown separately, the two organisms differed strongly with respect to the sterol composition. Sterols had a much higher relative abundance in the fungus in comparison with the plant, and the two main fungal sterols, ergosterol and 24-ethyllanosta-8,24(24′)-diene-3beta,22zeta-diol (Et lano 8,24), as well as six minor fungal sterols were not found in the plant. On the other hand, the three sterols found in plant roots were absent from the fungus. With regard to fatty acids, the lipids of both organisms contained the same three major PLFAs, namely n16:0, 18:2–9,12c, and 18:1–9c. However, plant lipids contained, in addition, eight PLFAs and five NLFAs that were not present in the fungus. On the other hand, the fungus contained two PLFAs and two NLFAs that were not present in the plant. When the fungus and the plant were brought together, there was a drastic change in the lipid composition of the root: within a day, all the saturated fatty acids in the NLFA fraction increased very strongly and then slowly decreased but remained at an elevated level throughout the experiment. All these saturated fatty acids also started to appear in the extraradical fungal mycelium; they increased steadily and reached their highest levels at the end of the experiment. These results indicate that in symbiosis, the fungus transports plant lipids from the symbiotic interface to the extraradical mycelium. Concerning sterols, the extraradical mycelium acquired only a small amount of plant-specific sterols. However, its ergosterol content steadily decreased whereas the content of Et lano 8,24 remained high, causing the ratio of these two sterols to decrease from 1 : 7 to 1 : 20, whereas in the ectomycorrhizal root, the opposite phenomenon occurred, so that the ratio increased to a value of almost 1 : 1. The marked changes in the composition of the extraradical mycelium were well reflected in a principal component analysis of all lipid components. The present results show that a given ectomycorrhizal fungus may display markedly different lipid compositions in its intraradical and extraradical parts. In addition, they highlight a potential role of plant lipid transfer from the root to the fungus in the functioning of the ectomycorrhizal symbiosis.