The main aim of this study was to test various hypotheses regarding the changes in δ13C of emitted CO2 that follow the addition of C4-sucrose to the soil of a C3-ecosystem. It forms part of an experimental series designed to assess whether or not the contributions from C3-respiration (root and microbial) and C4-respiration (microbial) to total soil respiration can be calculated from such changes. A series of five experiments, three on sieved (root-free) mor-layer material, and two in the field with intact mor-layer (and consequently with active roots), were performed. Both in the experiments on sieved mor-layer and the field experiments, we found a C4-sucrose-induced increase in C3-respiration that accounted for between 30% and 40% of the respiration increase 1 h after sucrose addition. When the course of C3-, C4- and total respiration was followed in sieved material over four days following addition of C4-sucrose, the initially increased respiration of C3-C was transient, passing within less than 24 h. In a separate pot experiment, neither ectomycorrhizal Pinus sylvestrisL. roots nor non-mycorrhizal roots of this species showed respiratory changes in response to exogenous sucrose. No shift in the δ13C of the evolved CO2 after adding C3-sucrose to sieved mor-layer material was found, confirming that the sucrose-induced increase in respiration of endogenous C was not an artefact of discrimination against 13C during respiration. Furthermore, we conclude that the C4-sucrose induced transient increase in C3-respiration is most likely the result of accelerated turnover of C in the microbial biomass. Thus, neither respiration of mycorrhizal roots, nor processes discriminating against δ13C were likely sources of error in the field. The estimated δ13C of evolved soil CO2 in three field experiments lay between −25.2‰ and −23.6‰. The study shows that we can distinguish between CO2 evolved from microbial mineralisation of added C4-sucrose, and CO2 evolved from endogenous carbon sources (roots and microbial respiration).