We tested whether 13C-discrimination during microbial respiration, or during CO2 sampling in the field, can explain changes observed in the δ13C of emitted CO2 that follow the addition of C4-sucrose, as a microbial substrate, to the soil of a C3-ecosystem. We approached this problem by adding C3-glucose (δ13C=–23.4‰), C4-sucrose (–10.8‰) or 13C-labelled glucose (103.7‰) to the intact mor layer, the upper organic soil (–26.5‰, bulk soil organic matter), of a boreal Pinus sylvestris L. forest. If 13C-discrimination is significant, it should generate illusory differences in the calculated contributions from the added C and endogenous C3-C to total soil respiration, when C4-sucrose or 13C-labelled glucose is added. Further, if discrimination occurs, we should also be able to detect a shift in the δ13C of respired CO2 after the addition of C3-glucose. The addition of the three sugar solutions gave similar increases in soil respiration (up to a doubling 1 h after the additions), while the addition of water gave no increase in respiration. There was no change in δ13C of the emitted CO2 after additions of H2O or C3-glucose. In contrast, the addition of C4-sucrose and 13C-labelled glucose gave δ13C values of evolved CO2 that were 4.5‰ and 30.3‰ higher than the pre-sugar values, respectively. The calculated respiration rates of the added carbon sources, C4-C or 13C-labelled C, were very similar. Also, we found very similar sugar-induced increases in respiration of endogenous C3-C in the plots supplied with C4-sucrose and 13C-labelled glucose, accounting for about 50% of the total increase in respiration 1 h after addition. Our results confirm that any microbial 13C-discrimination during respiration is minor.