The efficiency with which microbes use substrate (Carbon Use Efficiency or CUE) to make new microbial biomass is an important variable in soil and ecosystem C cycling models. It is generally assumed that CUE of microbial activity in soils is low, however measured values vary widely. It is hypothesized that high values of CUE observed in especially short-term incubations reflect the build-up of storage compounds in response to a sudden increase in substrate availability and are therefore not representative of CUE of microbial activity in unamended soil. To test this hypothesis, we measured the 13CO2 release from six position-specific 13C-labeled glucose isotopomers in ponderosa pine and pinon-juniper soil. We compared this position-specific CO2 production pattern with patterns expected for 1) balanced microbial growth (synthesis of all compounds needed to build new microbial cells) at a low, medium, or high CUE, and 2) synthesis of storage compounds (glycogen, tri-palmitoyl-glycerol, and polyhydroxybutyrate). Results of this study show that synthesis of storage compounds is not responsible for the observed high CUE. Instead, it is the position-specific CO2 production expected for balanced growth and high CUE that best matches the observed CO2 production pattern in these two soils. Comparison with published studies suggests that the amount of glucose added in this study is too low and the duration of the experiment too short to affect microbial metabolism. We conclude that the hypothesis of high CUE in undisturbed soil microbial communities remains viable and worthy of further testing.