Our understanding of forest biosphere–atmosphere interactions is fundamental for predicting forest ecosystem responses to climatic changes. Currently, however, our knowledge is incomplete partly due to inability to separate the major components of soil CO2 effluxes, viz. root respiration, microbial decomposition of soil organic matter and microbial decomposition of litter material. In this study we examined whether the δ13C characteristics of solid organic matter and respired CO2 from different soil-C components and root respiration in a Danish beech forest were useful to provide information on the root respiration contribution to total CO2 effluxes. The δ13C isotopic analyses of CO2 were performed using a FinniganMAT DeltaPLUS isotope-ratio mass spectrometer coupled in continuous flow mode to a trace gas preparation-concentration unit (PreCon). Gas samples in 2-mL crimp seal vials were analysed in a fully automatic mode with an experimental standard error ±0.11‰. We observed that the CO2 derived from root-free mineral soil horizons (A, BW) was more enriched in 13C (δ13C range −21.6 to −21.2‰) compared with CO2 derived from root-free humus layers (δ13C range −23.6 to −23.4‰). The CO2 evolved from root respiration in isolated young beech plants revealed a value intermediate between those for the soil humus and mineral horizons, δ13Croot = −22.2‰, but was associated with great variability (SE ± 1.0‰) due to plant-specific differences. δ13C of CO2 from in situ below-ground respiration averaged −22.8‰, intermediate between the values for the humus layer and root respiration, but variability was great (SE ± 0.4‰) due to pronounced spatial patterns. Overall, we were unable to statistically separate the CO2 of root respiration vs. soil organic matter decomposition based solely on δ13C signatures, yet the trend in the data suggests that root respiration contributed ∼43% to total respiration. The vertical gradient in δ13C, however, might be a useful tool in partitioning respiration in different soil layers. The experiment also showed an unexpected 13C-enrichment of CO2 (>3.5‰) compared with the total-C signatures in the individual soil-C components. This may suggest that analyses of bulk samples are not representative for the C-pools actively undergoing decomposition. Copyright © 2004 John Wiley & Sons, Ltd.