Rhizodeposits act as major carbon (C) source for microbial communities and rhizosphere-driven effects on forest C cycling receive increasing attention for maintaining soil biodiversity and ecosystem functions. By in situ 13CO2 pulse labeling we investigated C input and microbial utilization of rhizodeposits by analyzing 13C incorporation into phospholipid fatty acids (PLFA) of beech- (Fagus sylvatica) and ash-associated (Fraxinus excelsior) rhizomicrobial communities. Plant compartments and soil samples were analyzed to quantify the allocation of assimilates. For 1 m high trees, ash assimilated more of the applied 13CO2 (31%) than beech (21%), and ash allocated twice as much 13C belowground until day 20. Approximately 0.01% of the applied 13C was incorporated into total PLFAs, but incorporation varied significantly between microbial groups. Saprotrophic and ectomycorrhizal fungi under beech and ash, but also arbuscular mycorrhizal fungi and Gram negative bacteria under ash, incorporated most 13C. PLFA allowed differentiation of C fluxes from tree roots into mycorrhiza: twice as much 13C was incorporated into the fungal biomarker 18:2ω6,9 under beech than under ash. Within 5 days, 30% of the fungal PLFA-C was replaced by rhizodeposit-derived 13C under beech but only 10% under ash. None of the other microbial groups reached such high C replacement, suggesting direct C allocation via ectomycorrhizal symbioses dominates the C flux under beech. Based on 13CO2 labeling and 13C tracing in PLFA we conclude that ash allocated more C belowground and has faster microbial biomass turnover in the rhizosphere compared to beech. © 2017 Elsevier Masson SAS