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Accurate estimates of microbial carbon use efficiency (CUE) are required to predict how global change will impact microbially-mediated ecosystem functions such as organic matter decomposition. Multiple approaches are currently used to quantify CUE but the extent to which estimates reflect methodological variability is unknown. This limits our ability to apply or cross-compare published CUE values. Here we evaluated the performance of five methods in a single soil under standard conditions. The microbial response to three substrate amendment rates (0.0, 0.05, and 2.0 mg glucose-C g(-1) soil) was examined using: C-13 and O-18 isotope tracing approaches which estimate CUE based on substrate uptake and growth dynamics; calorespirometry which infers growth and CUE from metabolic heat and respiration rates; metabolic flux analysis where CUE is determined as the balance between biosynthesis and respiration using position-specific (CO2)-C-13 production of labeled glucose; and stoichiometric modeling which derives CUE from elemental ratios of microbial biomass, substrate, and exoenzyme activity. The CUE estimates we obtained differed by method and substrate concentration, ranging under in situ conditions from < 0.4 for the substrate-nonspecific methods that do not use C tracers (O-18, stoichiometric modeling) to > 0.6 for the substrate-specific methods that trace glucose use (C-13 method, calorespirometry, metabolic flux analysis). We explore the different aspects of microbial metabolism that each method captures and how this affects the interpretation of CUE estimates. We recommend that users consider the strengths and weaknesses of each method when choosing the technique that will best address their research needs.