The rate of organic matter turnover in soil is a critical component of the terrestrial carbon cycle and is frequently estimated from measurements of respiration. For estimates to be reliable requires that isotopically labelled substrate uptake into the soil microbial biomass and its subsequent mineralization occurs almost simultaneously (i.e. no time delay). Here we investigated this paradigm using glucose added to an agricultural soil. Immediately after collection from the field, various concentrations of 14C-labeled glucose (1 μM to 10 mM) were added to soil and the depletion from the soil solution measured at 1-60 min after substrate addition. 14CO2 production from the mineralization of glucose was simultaneously measured. The microbial uptake of glucose from soil solution was concentration-dependent and kinetic analysis suggests the operation of at least two distinct glucose transport systems of differing affinity. At glucose concentrations reflecting those naturally present in the soil solution (54±10 μM), the half-time (t1/2) of exogenous glucose was extremely rapid at ca. 30 s. At higher glucose concentrations (100 μM to 10 mM), the t1/2 values for the high-affinity carrier were altered little, but increasing proportions of glucose were taken up by the low affinity transport system. Glucose mineralization by the soil microbial community showed a significant delay after its uptake into the microbial biomass suggesting a decoupling of glucose uptake and subsequent respiration, possibly by dilution of glucose in labile metabolite pools. By fitting a double first order kinetic equation to the mineralization results we estimated the t1/2 for the first rapid phase of respiration at natural soil solution glucose concentrations to be 6-8 min, but at least 87% of the added glucose was retained in the microbial biomass prior to mineralization. Our results suggest that in this soil the soil solution glucose pool turns over 100-1000 times each day, an order of magnitude faster than when determined from measurements of mineralization. These results imply that traditional isotopic based measurements of substrate turnover measured using CO2 may vastly underestimate their rate of cycling in soil.