The main findings of research into carbon (C) fluxes from plants to soil micro-organisms using in situ 13CO2 pulse-labelling on upland grassland at the NERC Soil Biodiversity Thematic Programme field site in Southern Scotland are reviewed. From 1999 to 2003 the site was the focus of a unique and intensive programme of stable isotope tracing of C flux through rhizodeposition to soil microbiota and stable isotope probing of microbial biomarker compounds. We review the findings published to date, and highlight the novel ways in which the pulse-labelling approach has been applied to further understand C fluxes in the rhizosphere and mycorrhizophere in this grassland. The most important achievements from these studies, many of which are the first field measurements of their kind, include: (1) quantification of C flux from recent photosynthate into roots, soil microbial populations and soil respiration over time periods of hours to months; (2) analysis of diurnal control of root exudation and respiration linked to photoperiod and photosynthetic activity; (3) measurements of C flux from plants directed through mycorrhizal fungal networks; (4) establishing the importance C flow from recent photosynthate into soil fungi, revealed by 13C enrichment of phospholipid fatty acid biomarker molecules (PLFA); (5) detection of the disruptive effects of fungal-feeding microarthropods on 13CO2 respiration in the mycorrhizosphere; (6) measurement of 13C enrichment into soil microbial DNA and RNA and the rates of turnover of RNA; (7) identification of soil micro-organisms most enriched with 13C by sequence analysis of ‘heavy’ RNA separated by density-gradient centrifugation; and (8) estimates of the effects of liming on C flux into and through upland grassland, and its effects on C cycling by soil micro-organisms. In reviewing all these findings we highlight the strengths and limitations of the in situ 13C technique. We also explain how the new insights gained from these studies emphasise the complex temporal dynamics of recent photosynthate entering the soil through different pathways and the role of multi-trophic interactions between soil biota in determining the fate of recently fixed carbon in grasslands.