Photosynthetic carbon (C) isotope discrimination (ΔΑ) labels photosynthates (δA) and atmospheric CO2 (δa) with variable C isotope compositions during fluctuating environmental conditions. In this context, the C isotope composition of respired CO2 within ecosystems is often hypothesized to vary temporally with ΔΑ. We investigated the relationship between ΔΑ and the C isotope signals from stem (δW), soil (δS) and ecosystem (δE) respired CO2 to environmental fluctuations, using novel tuneable diode laser absorption spectrometer instrumentation in a mature maritime pine forest. Broad seasonal changes in ΔΑ were reflected in δW,δS and δE. However, respired CO2 signals had smaller short-term variations than ΔA and were offset and delayed by 2–10 d, indicating fractionation and isotopic mixing in a large C pool. Variations in δS did not follow ΔA at all times, especially during rainy periods and when there is a strong demand for C allocation above ground. It is likely that future isotope-enabled vegetation models will need to develop transfer functions that can account for these phenomena in order to interpret and predict the isotopic impact of biosphere gas exchange on the C isotope composition of atmospheric CO2.