Rhizodeposit-carbon provides a major energy source for microbial growth in the rhizosphere of grassland soils. However, little is known about the microbial communities that mediate the rhizosphere carbon dynamics, especially how their activity is influenced by changes in soil management. We combined a 13CO2 pulse-labeling experiment with phospholipid fatty acid (PLFA) analysis in differently managed Belgian grasslands to identify the active rhizodeposit-C assimilating microbial communities in these grasslands and to evaluate their response to management practices. Experimental treatments consisted of three mineral N fertilization levels (0, 225 and 450 kg N ha-1 y-1) and two mowing frequencies (3 and 5 times y-1). Phospholipid fatty acids were extracted from surface (0-5 cm) bulk (BU) and root-adhering (RA) soil samples prior to and 24 h after pulse-labeling and were analyzed by gas chromatography-combustion-isotope ratio mass spectrometry (GC-c-IRMS). Soil habitats significantly differed in microbial community structure (as revealed by multivariate analysis of mol% biomarker PLFAs) as well as in gram-positive bacterial rhizodeposit-C uptake (as revealed by greater 13C-PLFA enrichment following pulse-labeling in RA compared to BU soil in the 450N/5M treatment). Mowing frequency did not significantly alter the relative abundance (mol or activity (13C enrichment) of microbial communities. In the non-fertilized treatment, the greatest 13C enrichment was seen in all fungal biomarker PLFAs (C16:1?5, C18:1?9, C18:2? 6,9 and C18:3?3,6,9), which demonstrates a prominent contribution of fungi in the processing of new photosynthate-C in non-fertilized grassland soils. In all treatments, the lowest 13C enrichment was found in gram-positive bacterial and actinomycetes biomarker PLFAs. Fungal biomarker PLFAs had significantly lower 13C enrichment in the fertilized compared to non-fertilized treatments in BU soil (C16:1?5, C18:1?9) as well as RA soil (all fungal biomarkers). While these observations clearly indicated a negative effect of N fertilization on fungal assimilation of plant-derived C, the effect of N fertilization on fungal abundance could only be detected for the arbuscular mycorrhizal fungal (AMF) PLFA (C16:1?5). On the other hand, increases in the relative abundance of gram-positive bacterial PLFAs with N fertilization were found without concomitant increasesin 13C enrichment following pulse-labeling. We conclude that in situ 13C pulse-labeling of PLFAs is an effective tool to detect functional changes of those microbial communities that are dominantly involved in the immediate processing of new rhizosphere-C.