We combined microbial community phospholipid fatty acid (PLFA) analyses with an in situ stable isotope 1 3 CO 2 labelling approach to identify microbial groups actively involved in assimilation of root-derived C in limed grassland soils. We hypothesized that the application of lime would stimulate more rapid 1 3 C assimilation and turnover in microbial PLFAs. Four and 8d after label application, 18:1ω9, 18:2ω6,9 (fungal biomarkers) and 16:1ω7, 18:1ω7, 19:0cy (Gram-negative bacterial biomarkers) showed the most 1 3 C enrichment and rapid turnover rates. This suggests that these microorganisms were assimilating recently-photosynthesized root C inputs to soils. Contrary to our hypothesis, liming did not affect assimilation or turnover rates of 1 3 C-labelled C. 1 3 C stable isotope pulse-labelling technique paired with analyses of PLFA microbial biomarkers shows promise for in situ investigations of microbial function in soils.