We have studied how disturbance by ploughing and rotavation affects the carbon (C) flow to arbuscular mycorrhizal (AM) fungi in a dry, semi-natural grassland. AM fungal biomass was estimated using the indicator neutral lipid fatty acid (NLFA) 16:1ω5, and saprotrophic fungal biomass using NLFA 18:2ω6,9. We labeled vegetation plots with (13)CO(2) and studied the C flow to the signature fatty acids as well as uptake and allocation in plants. We found that AM fungal biomass in roots and soil decreased with disturbance, while saprotrophic fungal biomass in soil was not influenced by disturbance. Rotavation decreased the (13)C enrichment in NLFA 16:1ω5 in soil, but (13)C enrichment in the AM fungal indicator NLFA 16:1ω5 in roots or soil was not influenced by any other disturbance. In roots, (13)C enrichment was consistently higher in NLFA 16:1ω5 than in crude root material. Grasses (mainly Festuca brevipila) decreased as a result of disturbance, while non-mycorrhizal annual forbs increased. This decreases the potential for mycorrhizal C sequestration and may have been the main reason for the reduced mycorrhizal C allocation found in disturbed plots. Disturbance decreased the soil ammonium content but did not change the pH, nitrate or phosphate availability. The overall effect of disturbance on C allocation was that more of the C in AM fungal mycelium was directed to the external phase. Furthermore, the functional identity of the plants seemed to play a minor role in the C cycle as no differences were seen between different groups, although annuals contained less AM fungi than the other groups.