To study whether the biomass of soil microorganisms in a boreal Pinus sylvestris-Vaccinium vitis-idaea forest was limited by the availability of carbon or nitrogen, we applied sucrose from sugar cane, a C4 plant, to the organic mor-layer of the C3–C dominated soil. We can distinguish between microbial mineralization of the added sucrose and respiration of endogenous carbon (root and microbial) by using the C4-sucrose as a tracer, exploiting the difference in natural abundance of 13C between the added C4-sucrose (δ13C −10.8‰) and the endogenous C3–carbon (δ13C −26.6 ‰). In addition to sucrose, NH4Cl (340 kg N ha−1) was added factorially to the mor-layer. We followed the microbial activity for nine days after the treatments, by in situ sampling of CO2 evolved from the soil and mass spectrometric analyses of δ13C in the CO2. We found that microbial biomass was limited by the availability of carbon, rather than nitrogen availability, since there was a 50% increase in soil respiration in situ between 1 h and 5 days after adding the sucrose. However, no further increase was observed unless nitrogen was also added. Analyses of the δ13C ratios of the evolved CO2 showed that increases in respiration observed between 1 h and 9 days after the additions could be accounted for by an increase in mineralization of the added C4–C.