To assess how heterotrophic microorganisms may alter their activities and thus their CO2-C return to the atmosphere with elevated CO2 and changing N availability, we examined soil organic matter (SOM) dynamics at the Duke Free Air Carbon Enrichment (FACE) site, after N fertilizer was applied. We measured heterotrophic respiration during early and late stages of SOM mineralization in soil incubations to capture activity on relatively labile and refractory SOM pools. We also measured δ13C of respired CO2-C and phospholipid fatty acids (PLFAs) during early mineralization stages to track the microbial groups involved in substrate use. We calculated , a measure of δ13CPLFA normalized by respired δ13CO2, to assess microbial function with C substrates formed with elevated CO2 and altered N availability, via the distinct δ13C of the supplemental CO2. We also quantified extracellular enzyme activity (EEA) during labile and recalcitrant SOM mineralization. Early in the incubations, increased N availability reduced heterotrophic CO2-C release. By the later stages of SOM mineralization, elevated CO2 soils with fertilization had respired 72% of the CO2-C respired by all other soils. values suggest that fungi in elevated CO2 plots took up C substrates possessing the δ13C signature of recently formed SOM, and added N promoted the activity of Gram-negative bacteria and reduced that of Gram-positive bacteria, particularly actinomycetes. Consistent with this, the enzyme responsible for the degradation of peptidoglycan and chitin, compounds produced by Gram-positive bacteria and fungi, respectively, experienced a decline in activity with N fertilization. If patterns observed in this study with N additions are reversed with progressive N limitation at this site, actinomycetes and other Gram-positive bacteria responsible for mineralizing relatively recalcitrant substrates may experience increases in their activity. Such shifts in microbial functioning may result in increased turnover of, and C release from, relatively decay-resistant material.