In this study, we first determined the low molecular weight dissolved organic carbon (LMW DOC) concentration-dependent kinetics of soil respiration in a temperate grassland soil sampled on successive occasions. We then used the established relationship to estimate in situ LMW DOC concentrations from basal respiration measurements. 14C-labelled glucose was used as a model substrate and was added to soil over a wide range of concentrations (0.05–4000 μg C g−1 DW soil; equivalent to ca. 2.5 μM–200 mM glucose-C). The time-dependent loss of 14C-glucose to 14CO2 was similar to previous assessments. The Michaelis–Menten parameter Vmax varied between 17 (September 2010) and 42 (October 2010) μg CO2–C g−1 h−1 (corresponding to 1.4–3.5 µmol CO2 g−1 h−1), while Km varied between 893 (September 2010) and 1990 (October 2010) μg glucose-C g−1 (41–92 mM glucose), thus within the span previously reported for soils, albeit in the higher end of the range. However, the estimates were 6 orders-of-magnitude greater than those found in previous studies in natural waters. A possible methodological reason for this difference was an induced multiphasic concentration dependence, biasing Km and Vmax with high concentrations of LMW DOC. By combining the established concentration dependences with measurements of basal respiration, we estimated in situ concentrations of LMW DOC of 131 (October 2010), 112 (January 2011) and 270 (September 2010) μg LMW DOC g−1, far exceeding the total DOC concentration in the soil (17–20 μg DOC g−1 soil), thus invalidating our approach. We propose a way forward, and suggest that although current estimates of LMW DOC cycling need revision, there is evidence for a rapidly cycling pool of LMW DOC, possibly turning over >30 times per day, that warrants further attention.